CN116249556A - Antibody conjugation chemical inducer for degrading BRM and method thereof - Google Patents

Antibody conjugation chemical inducer for degrading BRM and method thereof Download PDF

Info

Publication number
CN116249556A
CN116249556A CN202180060539.4A CN202180060539A CN116249556A CN 116249556 A CN116249556 A CN 116249556A CN 202180060539 A CN202180060539 A CN 202180060539A CN 116249556 A CN116249556 A CN 116249556A
Authority
CN
China
Prior art keywords
conjugate
cide
antibody
group
brm
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202180060539.4A
Other languages
Chinese (zh)
Inventor
P·S·德拉格维奇
S·A·贝克·多克雷
T·H·皮洛
D·张
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genentech Inc
Original Assignee
Genentech Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genentech Inc filed Critical Genentech Inc
Publication of CN116249556A publication Critical patent/CN116249556A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Abstract

The subject matter described herein relates to antibody-CIDE conjugates (Ab-CIDE) that target BRMs for degradation, pharmaceutical compositions comprising them, and their use in treating diseases and conditions where BRM degradation is beneficial.

Description

Antibody conjugation chemical inducer for degrading BRM and method thereof
Cross Reference to Related Applications
The present application claims priority and benefit from U.S. patent application No. 63/054,757, filed 7/21/2020, the disclosure of which is incorporated herein by reference in its entirety.
Sequence listing
The formal copy of the sequence list is submitted electronically in the form of an ASCII format sequence list via the EFS-Web, with a file name of P36010-WO_SL.txt, created at 20 days 7 of 2021, and a size of 44,936 bytes, and submitted concurrently with the description. The sequence listing contained in this ASCII formatted document is part of the specification, the entire contents of which are incorporated herein by reference.
Technical Field
The subject matter described herein relates generally to degradant conjugates comprising antibody-proteolysis-targeting chimeric molecules that can be used to promote intracellular degradation of a target BRM protein.
Background
Cell maintenance and normal function require controlled degradation of cellular proteins. For example, degradation of regulatory proteins can trigger events in the cell cycle, such as DNA replication, chromosome segregation, and the like. Thus, this protein degradation affects cell proliferation, differentiation and death.
Although protein inhibitors may prevent or reduce the activity of a protein in a cell, protein degradation in a cell may also reduce the activity or completely remove a target protein. Thus, the protein degradation pathway utilizing cells may provide a means to reduce or eliminate protein activity. One of the major degradation pathways of cells is known as the ubiquitin-proteasome system. In this system, the protein is labeled by ubiquitination of the protein, which is degraded by the proteasome. Ubiquitination of proteins is accomplished by an E3 ubiquitin ligase that binds the protein and adds ubiquitin molecules to the protein. The E3 ubiquitin ligase is part of a pathway that includes E1 and E2 ubiquitin ligases, which allows the E3 ubiquitin ligase to add ubiquitin to proteins.
To exploit this degradation pathway, a molecular structure called chemical degradation inducer (CIDE) binds E3 ubiquitin ligase with the protein to be degraded. To facilitate proteasome degradation of proteins, CIDE includes a group that binds to the E3 ubiquitin ligase and a group that binds to the protein target to be degraded. These groups are typically attached to a linker. The molecular CIDE allows the E3 ubiquitin ligase to be brought into proximity with the protein, thereby ubiquitinating it and labeling it for degradation. However, the relatively large size of CIDE can cause problems for targeted delivery and can lead to undesirable properties such as rapid metabolism/clearance, short half-life and low bioavailability.
There is a continuing need in the art for improved CIDE, including enhanced targeted delivery of CIDE to cells comprising protein targets. The subject matter described herein addresses this and other shortcomings in the art.
Disclosure of Invention
In one aspect, the subject matter described herein relates to conjugated or covalently linked Ab-CIDE, wherein the position of the covalent bond linking the following components of the Ab-CIDE: the antibody (Ab), linker 1 (L1), linker 2 (L2), protein binding group (PB) and E3 ligase binding group (E3 LB) can be tailored as desired to produce Ab-CIDE with desired properties such as potency, in vivo pharmacokinetics, stability and solubility.
In one aspect, the subject matter described herein relates to Ab-CIDE having the following chemical structure:
Ab-(L1-D) p ，
wherein ,
ab is an antibody;
d is CIDE or prodrug thereof, having the following structure:
Figure BDA0004113777320000021
wherein ,
BRM is the residue of a BRM binding compound,
e3LB is the residue of an E3 ligase binding compound, and
l2 is a moiety that covalently links BRM and E3 LB;
l1 is linker-1 covalently linking Ab to one of BRM, E3LB, or L2; and is also provided with
p is 1 to 16.
In another aspect, the subject matter described herein relates to Ab-CIDE having the following chemical structure:
Ab-(L1-D) p ，
wherein ,
ab is an antibody;
D is CIDE or prodrug thereof, having the following structure:
Figure BDA0004113777320000031
wherein L1 is attached at one attachment point selected from the group consisting of: L1-Q, L-Q', L1-S,
L1-T and optionally L1-U, L1-V and L1-Y, if present, wherein
L1Q on BRM
Figure BDA0004113777320000032
Where M is O;
L1-Q' on BRM
Figure BDA0004113777320000033
Where M' is-NH;
L1-S on L2
Figure BDA0004113777320000034
Figure BDA0004113777320000035
A place; />
L1-T on E3LB
Figure BDA0004113777320000036
Wherein A is a group covalently bonded to L2;
L1-U and L1-V on E3LB
Figure BDA0004113777320000041
A place; and is also provided with
L1-Y on E3LB
Figure BDA0004113777320000042
Figure BDA0004113777320000043
Where, there is->
Figure BDA0004113777320000046
Is a single bond or a double bond.
In another aspect, the subject matter described herein relates to Ab-CIDE having the following chemical structure:
Ab-(L1-D) p ，
wherein ,
ab is an antibody;
d is CIDE or prodrug thereof, having the following structure:
Figure BDA0004113777320000044
wherein ：
R 3 is cyano group,
Figure BDA0004113777320000045
wherein ,
Figure BDA0004113777320000053
is a single bond or a double bond.
In another aspect, the subject matter described herein relates to Ab-CIDE having the following chemical structure:
Ab-(L1-D) p ，
wherein ,
ab is an antibody;
d is CIDE or prodrug thereof, having the following structure:
Figure BDA0004113777320000051
wherein ,R1A 、R 1B and R1C Each independently is hydrogen or C 1-5 An alkyl group; or R is 1A 、R 1B and R1C Two of which together with the carbon to which each is attached form C 1-5 Cycloalkyl groups.
In another aspect, the subject matter described herein relates to Ab-CIDE having the following chemical structure:
Ab―(L1―D) p ，
wherein ,
d is CIDE with E3 LB-L2-PB structure;
e3LB is covalently bound to L2, said E3LB having the formula:
Figure BDA0004113777320000052
wherein ,
R 1A 、R 1B and R1C Each independently is hydrogen or C 1-5 An alkyl group; or R is 1A 、R 1B And
R 1C two of which together with the carbon to which each is attached form C 1-5 Cycloalkyl;
R 2 is C 1-5 An alkyl group;
R 3 selected from the group consisting of cyano groups,
Figure BDA0004113777320000061
A group of (I), wherein->
Figure BDA0004113777320000065
Is a single bond or a double bond;
Y 1 and Y2 One of them is-CH, Y 1 and Y2 The other of them is-CH or N;
l2 is a linker covalently bound to E3LB and PB, said L2 having the formula:
Figure BDA0004113777320000062
wherein ,
R 4 is hydrogen or methyl, and is preferably hydrogen or methyl,
Figure BDA0004113777320000063
wherein ,
z is one or zero and is zero,
g is
Figure BDA0004113777320000064
or-C (O) NH-; and is also provided with
Figure BDA0004113777320000071
Is a connection point with PB
PB is a protein binding group covalently bound to L2, which has the following structure:
Figure BDA0004113777320000072
ab is an antibody covalently bound to at least one L1, L1 being a linker;
L1-T, L1-U and L1-V are each independently hydrogen or an L1 linker covalently bound to Ab and D;
L1-Y is hydrogen or an L1 linker covalently bound to Ab and D;
q is 1 or 0;
and ,
p has a value of about 1 to about 8.
Another aspect of the subject matter described herein is a pharmaceutical composition comprising Ab-CIDE and one or more pharmaceutically acceptable excipients.
Another aspect of the subject matter described herein is the use of Ab-CIDE in a method of treating symptoms and diseases by administering to a subject a pharmaceutical composition comprising Ab-CIDE.
Another aspect of the subject matter described herein is a method of making Ab-CIDE.
Another aspect of the subject matter described herein is an article of manufacture comprising a pharmaceutical composition comprising Ab-CIDE, a container, and a package insert or label page indicating that the pharmaceutical composition is useful for treating a disease or condition.
Other embodiments are also fully described herein.
Drawings
FIGS. 1A and 1B show that exemplary Ab-CIDE Ab-L1A-CIDE-BRM1-1 has activity in a cellular level assay.
FIGS. 2A and 2B show that exemplary Ab-CIDE Ab-L1a-CIDE-BRM1-3 have activity in cell level assays.
FIGS. 3A-3L show the dose and antigen-dependent antitumor activity of exemplary Ab-CIDE Ab-L1a-CIDE-BRM 1-1.
FIGS. 4A-4L show the dose and antigen-dependent antitumor activity of exemplary Ab-CIDE Ab-L1a-CIDE-BRM 1-3. The data are compared with the data of Ab-CIDE Ab-L1a-CIDE-BRM 1-1.
FIG. 5 shows that BRM and BRG1 degradation correlates with anti-tumor activity of exemplary Ab-CIDE Ab-L1a-CIDE-BRM 1-1.
FIG. 6 shows that BRM and BRG1 degradation is less correlated with anti-tumor activity of exemplary Ab-CIDE Ab-L1a-CIDE-BRM 1-3. All channels were used for Ab-CIDE Ab-L1a-CIDE-BRM1-3, but the expression "channels" were used for Ab-CIDE-L1a-BRM 1-1.
Figure 7 shows that antibody ligation strategy can modulate the activity of CIDE. The data shows that while CIDE-BRM1-3 is generally more effective than CIDE-BRM1-1, exemplary Ab-CIDE Ab-L1a-CIDE-BRM1-1 provides stronger BRM degradation than unconjugated CIDE-BRM 1-3. All channels were used for Ab-CIDE Ab-L1a-CIDE-BRM1-1, but the expression "channels" were used for Ab-CIDE-L1a-BRM 1-3.
Figures 8-12 depict some antibody ligation strategies described herein.
Detailed Description
Disclosed herein are antibody-chemical degradation inducer ("CIDE") conjugates, referred to herein as Ab-CIDE, that are useful for targeted protein degradation of BRM (also known as SMARCA 2) and treatment of related diseases and conditions. In particular, the disclosure relates to a CIDE conjugated to an antibody comprising a ligand that binds to Von Hippel-Lindau E3 ubiquitin ligase at one end and a moiety that binds to BRM (target protein) at the other end such that the target protein is placed in proximity to ubiquitin ligase to effect degradation, thereby modulating BRM. The connection strategy and type of the linker is adjusted as described herein, and the reported data shows that such adjustment can have a beneficial effect on the activity of CIDE on BRM.
The subject matter described herein utilizes antibody targeting to direct CIDE to a target cell or tissue. As described herein, it has been shown that linking antibodies to the CIDE to form Ab-CIDE can deliver the CIDE to a target cell or tissue. As shown herein, for example, in an embodiment, cells expressing an antigen can be targeted by an antigen-specific Ab-CIDE, whereby the CIDE moiety of the Ab-CIDE is delivered intracellularly to the target cell. CIDE comprising antibodies to antigens not found on cells does not result in substantial intracellular delivery of CIDE to the cells.
Accordingly, the subject matter described herein relates to Ab-CIDE compositions that result in ubiquitination of target proteins and subsequent protein degradation. The composition comprises an antibody covalently linked to linker 1 (L1) which is covalently linked to a CIDE at any available point of attachment, wherein the CIDE comprises an E3 ubiquitin ligase binding (E3 LB) moiety, wherein the E3LB moiety recognizes an E3 ubiquitin ligase protein (VHL), and linker 2 (L2) covalently links the E3LB moiety to a protein binding moiety (PB) which is a moiety that recognizes a target protein BRM or SMARCA 2. The subject matter described herein is useful for degrading and thus modulating protein activity, and treating diseases and conditions associated with protein activity.
The presently disclosed subject matter will now be described more fully hereinafter. However, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. In other words, the subject matter described herein encompasses all alternatives, modifications, and equivalents. If one or more of the incorporated documents, patents and similar materials is different or contradicted by this application, including but not limited to defined terms, use of terms, described techniques or the like, this application controls. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.
I. Definition of the definition
The term "CIDE" refers to a chemical degradation inducer that is a proteolytically targeted chimeric molecule, typically having the following three components: an E3 ubiquitin ligase binding group (E3 LB), a linker L2 and a protein binding group (PB).
The term "residue", "moiety", "constituent" or "group" refers to a component that is covalently bound or linked to another component. The term "component" is also used herein to describe such residues, moieties, components or groups. For example, a residue of a compound will have one or more atoms of the compound (e.g., hydrogen or hydroxyl) covalently substituted, thereby binding the residue to CIDE, L1-CIDE, or another component of Ab-CIDE. For example, a "residue of CIDE" refers to a CIDE covalently linked to one or more groups, such as linker L2, which may itself optionally be further linked to an antibody.
The term "covalently bound" or "covalently linked" refers to a chemical bond formed by sharing one or more pairs of electrons.
As used herein, the term "peptidomimetic" or PM refers to a non-peptide chemical moiety. Peptides are short chains of amino acid monomers linked by peptide (amide) bonds, i.e., covalent chemical bonds that are formed when the carboxyl group of one amino acid reacts with the amino group of another amino acid. The shortest peptide is a dipeptide (consisting of 2 amino acids linked by a single peptide bond), followed by a tripeptide, tetrapeptide, etc. The peptidomimetic chemical moiety includes a non-amino acid chemical moiety. The peptidomimetic chemical moiety can also comprise one or more amino acids separated by one or more non-amino acid-containing units. The peptidomimetic chemical moiety does not comprise two or more adjacent amino acids linked by peptide bonds in any portion of its chemical structure.
The term "antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity (Miller et al (2003) journal of Immunology 170:4854-4861). The antibody may be a murine antibody, a human antibody, a humanized antibody, a chimeric antibody or an antibody derived from another species. Antibodies are proteins produced by the immune system that are capable of recognizing and binding to a specific antigen. (Janeway, c., convers, p., walport, m., shomchik (2001) immune Biology, 5 th edition, garland Publishing, new York). The target antigen typically has multiple binding sites, also known as epitopes, recognized by CDRs (complementarity determining regions) on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, an antigen may have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to an antigen or portion thereof of a target of interest, including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein can be of any type (e.g., igG, igE, igM, igD and IgA), class (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2) or subclass of immunoglobulin molecule. The immunoglobulin may be derived from any species. However, in one aspect, the immunoglobulin is of human, murine or rabbit origin.
As used herein, the term "antibody fragment" includes a portion of a full-length antibody, typically the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, fab ', F (ab') 2 And Fv fragments; a diabody antibody; a linear antibody;minibodies (Olafsen et al (2004) Protein Eng. Design)&Sel.17 (4): 315-323), fragments generated from Fab expression libraries, anti-idiotype (anti-Id) antibodies, CDRs (complementarity determining regions) and epitope-binding fragments of any of the above, which immunospecifically bind to a cancer cell antigen, viral antigen or microbial antigen, single chain antibody molecules; and multispecific antibodies formed from antibody fragments.
The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprised by the population are identical except for minor naturally occurring mutations that may be present. Monoclonal antibodies have a high specificity for a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to specificity, monoclonal antibodies are advantageous in that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the subject matter described herein may be prepared by the hybridoma method described first by Kohler et al (1975) Nature,256:495, or may be prepared by recombinant DNA methods (see, e.g., US 481757, US 5807715). "monoclonal antibodies" can also be isolated from phage antibody libraries using, for example, the following references: clackson et al (1991) Nature,352:624-628; marks et al (1991) J.mol.biol.,222:581-597.
Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of one or more chains is identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided that they exhibit the desired biological activity (US 4815567; and Morrison et al (1984) Proc.Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen binding sequences derived from a non-human primate (e.g., old world monkey, ape, etc.), as well as human constant region sequences.
The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chains are derived from a particular source or species, while the remainder of the heavy and/or light chains are derived from a different source or species.
The "class" of antibodies refers to the type of constant domain or constant region that the heavy chain of an antibody has. There are five main classes of antibodies: igA, igD, igE, igG and IgM, and some of them may be further classified into subclasses (isotypes), for example, igG 1 、IgG 2 、IgG 3 、IgG 4 、IgA 1, and IgA2 . The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively.
As used herein, the term "whole antibody" is an antibody comprising VL and VH domains and light chain constant domain (CL) and heavy chain constant domains CH1, CH2 and CH 3. The constant domain may be a natural sequence constant domain (e.g., a human natural sequence constant domain) or an amino acid sequence variant thereof. An intact antibody may have one or more "effector functions," which refer to those biological activities attributable to the Fc constant region (native sequence Fc region or amino acid sequence variant Fc region) of the antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down-regulating cell surface receptors, such as B cell receptors and BCR.
As used herein, the term "Fc region" means the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxy terminus of the heavy chain. However, the C-terminal lysine (Lys 447) of the Fc region may or may not be present. Unless otherwise indicated herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described by Kabat et al (Sequences of Proteins of Immunological Interest, 5 th edition, U.S. department of health and public service, national institutes of health, besseda, 1991).
As used herein, the term "framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of the variable domain typically consists of four FR domains: FR1, FR2, FR3 and FR4. Thus, HVR and FR sequences typically occur in VH (or VL) with the following sequences: FR1-H1 (L1) -FR2-H2 (L2) -FR3-H3 (L3) -FR4.
The terms "full length antibody", "whole antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to the structure of a natural antibody or having a heavy chain comprising an Fc region as defined herein.
A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or an amino acid sequence derived from a non-human antibody that utilizes a repertoire of human antibodies or other human antibody coding sequences. This definition of human antibodies specifically excludes humanized antibodies that comprise non-human antigen binding residues.
"humanized" antibody refers to chimeric antibodies that comprise amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. An antibody in a "humanized form", e.g., a non-human antibody, refers to an antibody that has been humanized.
An "isolated antibody" is an antibody that has been isolated from a component of its natural environment. In some embodiments, the antibodies are purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (isoelectric focusing, IEF), capillary electrophoresis), or chromatography (e.g., ion exchange or reverse phase HPLC). For reviews of methods of assessing antibody purity, see, e.g., flatman et al, J.chromatogrB 848:79-87 (2007).
An "isolated nucleic acid" refers to a nucleic acid molecule that has been separated from components of its natural environment. Isolated nucleic acids include nucleic acid molecules that are contained in cells that normally contain the nucleic acid molecule, but which are present extrachromosomally or at a chromosomal location different from that of their natural chromosome location.
"isolated nucleic acid encoding an antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of the antibody, including such nucleic acid molecules in a single vector or in separate vectors, as well as such nucleic acid molecules present at one or more positions in a host cell.
"naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabeled. The naked antibody may be present in a pharmaceutical formulation.
"Natural antibody" refers to naturally occurring immunoglobulin molecules having different structures. For example, a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH 1, CH2 and CH 3). Similarly, from N-terminal to C-terminal, each light chain has a variable region (VL), also known as a variable light chain domain or light chain variable domain, followed by a constant light Chain (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa (kappa) and lambda (lambda), based on the amino acid sequence of its constant domain.
"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after aligning the candidate sequence to the reference polypeptide sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without regard to any conservative substitutions as part of the sequence identity. The alignment used to determine the percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences compared. However, for purposes herein, the sequence comparison computer program ALIGN-2 was used to generate values for% amino acid sequence identity. ALIGN-2 sequence comparison computer programs were written by Genntech, inc., and the source code had been submitted with the user document to U.S. Copyright Office, washington D.C.,20559, where it was registered with U.S. copyright accession number TXU 510087. The ALIGN-2 program is publicly available from Genntech, inc. (Inc., south San Francisco, california) or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including the digital UNIX V4.0D. All sequence comparison parameters were set by the ALIGN-2 program and were unchanged.
In the case of amino acid sequence comparison using ALIGN-2, the amino acid sequence identity of a given amino acid sequence A with a given amino acid sequence B (which may alternatively be expressed as having or comprising some amino acid sequence identity with a given amino acid sequence B) is calculated as follows:
100 times the fraction X/Y
Wherein X is the number of amino acid residues scored as identical matches in the program alignment of A and B by the sequence alignment program ALIGN-2, and wherein Y is the total number of amino acid residues in B. It will be appreciated that in the case where the length of amino acid sequence a is not equal to the length of amino acid sequence B, the% amino acid sequence identity of a to B will not be equal to the% amino acid sequence identity of B to a. All values of% amino acid sequence identity as used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph, unless specifically indicated otherwise.
Complete antibodies can be classified into different "classes" according to the amino acid sequence of their heavy chain constant domains. There are five main classes of intact immunoglobulin antibodies: igA, igD, igE, igG and IgM, and several of these classes can be further divided into "subclasses" (isotypes), such as IgG1, igG2, igG3, igG4, igA, and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called α, δ, ε, γ and μ, respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Ig forms include hinge modified or non-hinge forms (Roux et al (1998) J.Immunol.161:4083-4090; lund et al (2000) Eur. J.biochem.267:7246-7256; US 2005/0048572; US 2004/0229310).
As used herein, the term "human consensus framework" refers to a framework that represents the amino acid residues most commonly present in the selection of human immunoglobulin VL or VH framework sequences. In general, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. In general, a subset of sequences is as described in Kabat et al, sequences of Proteins of Immunological Interest, 5 th edition, NIH Publication 91-3242, bethesda MD (1991), volumes 1-3. In one embodiment, for VL, the subgroup is subgroup κI as in Kabat et al, supra. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al, supra.
For purposes herein, a "recipient human framework" is a framework comprising an amino acid sequence derived from a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework of a human immunoglobulin framework or a human consensus framework as defined below. The recipient human framework "derived from" a human immunoglobulin framework or human consensus framework may comprise the same amino acid sequence as the human immunoglobulin framework or human consensus framework, or it may comprise amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.
As used herein, the term "variable region" or "variable domain" refers to a domain of an antibody heavy or light chain that is involved in binding an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved Framework Regions (FR) and three hypervariable regions (HVR). (see, e.g., kit et al Kuby Immunology, 6 th edition, w.h. freeman and co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind a particular antigen can be isolated using VH or VL domains, respectively, from antibodies that bind that antigen to screen libraries of complementary VL or VH domains. See, for example, portolano et al, J.Immunol.150:880-887 (1993); clarkson et al Nature 352:624-628 (1991).
As used herein, the term "hypervariable region" or "HVR" refers to any one of the antibody variable domain regions that are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops"). Typically, a natural four-chain antibody comprises six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and/or from "complementarity determining regions" (CDRs) which have the highest sequence variability and/or are involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J.mol. Biol.196:901-917 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3) occur at amino acid residues 24-34 of L1, amino acid residues 50-56 of L2, amino acid residues 89-97 of L3, amino acid residues 50-65 of amino acid residues 31-35B, H2 of H1, and amino acid residues 95-102 of H3. (Kabat et al, sequences of Proteins of Immunological Interest, 5 th edition, U.S. department of health and public service, national institutes of health, besselda (1991)) in Malyland. In addition to CDR1 in VH, CDRs typically comprise amino acid residues that form hypervariable loops. CDRs also contain "specificity determining residues" or "SDRs," which are residues that contact an antigen. SDR is contained within CDR regions known as shortened CDRs or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2 and a-CDR-H3) occur at amino acid residues 31-34 of L1, amino acid residues 50-55 of L2, amino acid residues 89-96 of L3, amino acid residues 31-35B, H of H1, amino acid residues 50-58 of H3, and amino acid residues 95-102. (see Almagro and Franson, front. Biosci.13:1619-1633 (2008)). Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to Kabat et al.
"effector functions" refer to those biological activities attributable to the Fc region of an antibody that vary with the variation of the antibody isotype. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); b cell activation.
The term "epitope" refers to a specific site on an antigen molecule to which an antibody binds.
"affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibodies and antigens). The affinity of a molecule X for its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below. In certain embodiments, the antibodies described herein have a dissociation constant (Kd) of 1. Mu.M, 100nM, 10nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.1nM, 0.01nM or 0.001nM (e.g., 10) -8 M or less, e.g. 10 -8 M to 10 -13 M, e.g. 10 -9 M to 10 -13 M)。
An "affinity matured" antibody refers to an antibody having one or more alterations in one or more hypervariable regions (HVRs) that result in an improvement in the affinity of the antibody for an antigen as compared to a parent antibody that does not have such alterations.
As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid linked thereto. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".
As used herein, the term "free cysteine amino acid" refers to a cysteine amino acid residue that has been engineered into a parent antibody, has a thiol functional group (-SH), and does not pair into an intramolecular or intermolecular disulfide bond. As used herein, the term "amino acid" refers to glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, serine, threonine, tyrosine, cysteine, methionine, lysine, arginine, histidine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine or citrulline.
As used herein, the term "linker," "linker unit," or "linker" refers to a chemical moiety comprising an atomic chain that covalently links a moiety of a CIDE to an antibody or a residue, moiety, group, or component of a CIDE to another residue, moiety, group, or component of a CIDE. In various embodiments, the linker is a divalent group, designated linker 1, linker 2, L1, or L2.
A "patient" or "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the patient or individual or subject is a human. In some embodiments, the patient may be a "cancer patient," i.e., a patient suffering from or at risk of suffering from one or more symptoms of cancer.
By "patient population" is meant a group of cancer patients. Such populations may be used to demonstrate statistically significant drug efficacy and/or safety.
The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is often characterized by uncontrolled cell growth/proliferation. A "tumor" comprises one or more cancer cells. Examples of cancers are provided elsewhere herein.
As used herein, "treatment" (and grammatical variations thereof, such as "treatment" or "treatment") refers to a clinical intervention that attempts to alter the natural course of the treated individual, and may be performed for prophylaxis or in the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of a disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating a disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the subject matter described herein are used to delay the progression of a disease or slow the progression of a disease.
A drug administered "concurrently" with one or more other drugs is administered on the same day of treatment with the one or more other drugs, and optionally concurrently with the one or more other drugs, within the same treatment cycle. For example, for cancer treatment once every 3 weeks, each drug administered simultaneously is administered on day 1 of the 3 week cycle.
An "effective amount" of an agent (e.g., a pharmaceutical formulation) refers to an amount effective to achieve a desired therapeutic or prophylactic result at the necessary dosage and time period. For example, a therapeutically effective amount of the drug may reduce the number of cancer cells; reducing tumor size; inhibit (i.e., slow and preferably stop to some extent) cancer cell infiltration of surrounding organs; inhibit (i.e., slow and preferably stop to some extent) tumor metastasis; inhibit tumor growth to some extent; and/or to some extent, alleviate one or more symptoms associated with cancer. To some extent, the drug may prevent growth and/or kill existing cancer cells, which may inhibit cell growth and/or be cytotoxic. An effective amount may extend progression free survival (e.g., as measured by a response assessment criterion for solid tumors, RECIST or CA-125 change), result in an objective response (including partial response, PR or complete response, CR), increase overall survival time, and/or improve one or more symptoms of cancer (e.g., as assessed by FOSI).
As used herein, the term "therapeutically effective amount" refers to any amount that results in the treatment of a disease, disorder, or side effect, or reduces the rate of progression of a disease or disorder, as compared to a corresponding subject that does not receive the amount. The term also includes within its scope an amount effective to enhance normal physiological function. For use in therapy, a therapeutically effective amount of Ab-CIDE and salts thereof may be administered as a raw chemical. In addition, the active ingredient may be present as a pharmaceutical composition.
The term "pharmaceutical formulation" refers to a formulation that is in a form that allows for the biological activity of the active ingredient contained therein to be effective, and that is free of additional components that have unacceptable toxicity to the subject to whom the formulation is to be administered.
"pharmaceutically acceptable excipient" refers to a component of a pharmaceutical formulation that is not toxic to a subject, except for the active ingredient. Pharmaceutically acceptable excipients include, but are not limited to, buffers, carriers, stabilizers or preservatives.
As used herein, the phrase "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a molecule. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, hydrochloride, bromate, iodate, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, gluconate, formate, benzoate, glutamate, mesylate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e., 1' -methylene-bis- (2-hydroxy-3-naphthoic acid)) salts. The pharmaceutically acceptable salt may include a composition comprising another molecule, such as an acetate ion, a succinate ion, or other counterion. The counterion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, the pharmaceutically acceptable salts may have more than one charged atom in their structure. Examples where multiple charged atoms are part of a pharmaceutically acceptable salt may have multiple counter ions. Thus, pharmaceutically acceptable salts may have one or more charged atoms and/or one or more counter ions.
Other non-pharmaceutically acceptable salts may be used in the preparation of the compounds described herein and should be considered as forming a further aspect of the subject matter. These salts, such as oxalic acid or trifluoroacetate salts, while not pharmaceutically acceptable per se, may be used to prepare salts useful as intermediates for obtaining the compounds described herein and pharmaceutically acceptable salts thereof.
As used herein, the term "alkyl" refers to a saturated straight or branched monovalent hydrocarbon radical of any length having one to five carbon atoms (C 1 -C 5 ) Wherein the alkyl groups may be optionally substituted independently with one or more substituents described below. In another embodiment, alkyl is one, two, three, four, or five carbon atoms. Examples of alkyl groups include, but are not limited to, methyl (Me, -CH 3 ) Ethyl (Et, -CH) 2 CH 3 ) 1-propyl (n-Pr, n-propyl, -CH) 2 CH 2 CH 3 ) 2-propyl (i-Pr, isopropyl, -CH (CH) 3 ) 2 ) 1-butyl (n-Bu, n-butyl, -CH) 2 CH 2 CH 2 CH 3 ) 2-methyl-1-propyl (i-Bu, isobutyl, -CH) 2 CH(CH 3 ) 2 ) 2-butyl (s-Bu, secondary butyl, -CH (CH) 3 )CH 2 CH 3 ) 2-methyl-2-propyl (t-Bu, tert-butyl, -C (CH) 3 ) 3 ) 1-pentyl (n-pentyl, -CH) 2 CH 2 CH 2 CH 2 CH 3 ) 2-pentyl (-CH (CH) 3 )CH 2 CH 2 CH 3 ) 3-pentyl (-CH (CH) 2 CH 3 ) 2 ) 2-methyl-2-butyl (-C (CH) 3 ) 2 CH 2 CH 3 ) 3-methyl-2-butyl (-CH (CH) 3 )CH(CH 3 ) 2 ) 3-methyl-1-butyl (-CH) 2 CH 2 CH(CH 3 ) 2 ) 2-methyl-1-butyl (-CH) 2 CH(CH 3 )CH 2 CH 3 ) Etc.
As used herein, the term "alkylene" refers to a saturated straight or branched divalent hydrocarbon radical of any length having one to twelve carbon atoms (C 1 -C 12 ) Wherein the alkylene groups may be optionally independently substituted with one or more substituents described below. In another embodiment, the alkylene group contains one to eight carbon atoms (C 1 -C 8 ) Or one to six carbon atoms (C 1 -C 6 ). Examples of alkylene groups include, but are not limited to, methylene (-CH) 2 (-), ethylene (-CH) 2 CH 2 (-), propylene (-CH) 2 CH 2 CH 2 (-), etc.
The terms "carbocycle", "carbocyclyl", "carbocycle" and "cycloalkyl" refer to a compound having 3 to 5 carbon atoms (C 3 -C 5 ) A monovalent non-aromatic, saturated or partially unsaturated ring. Examples of monocyclic carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, and the like. The carbocyclyl groups may be optionally substituted independently with one or more alkyl groups.
"heterocycle", "heterocyclic", "heterocycloalkyl" or "heterocyclyl" refers to a saturated or partially unsaturated group having a single ring or multiple condensed rings, including fused, bridged or spiro ring systems, and having from 3 to 20 ring atoms, including from 1 to 10 heteroatoms. These ring atoms are selected from the group consisting of carbon, nitrogen, sulfur, or oxygen, wherein in the fused ring system one or more of the rings may be cycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through a non-aromatic ring. In certain embodiments, the nitrogen and/or sulfur atoms of the heterocyclic group are optionally oxidized to provide an N-oxide, -S (O) -or-SO 2 -a portion. Examples of heterocycles include, but are not limited to, azetidine, indoline, indazole, quinolizine, imidazolidine, imidazoline, piperidine, piperazine, indoline, 1,2,3, 4-tetrahydroisoquinoline, thiazolidine, morpholinyl (also known as thiomorpholinyl), 1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like. Heterocyclyl groups can be asSubstituted as described in WO 2014/100762.
The term "chiral" refers to a molecule that has no overlap with a mirror partner, while the term "achiral" refers to a molecule that can overlap with its mirror partner.
The term "stereoisomers" refers to compounds having the same chemical composition but different arrangements of atoms or groups in space.
"diastereoisomers" means stereoisomers which have two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral characteristics, and reactivity. Mixtures of diastereomers can be separated under high resolution analytical procedures such as electrophoresis and chromatography.
"enantiomer" refers to two stereoisomers of a compound that are mirror images of each other that are non-superimposable.
Stereochemical definitions and conventions used herein generally follow: S.P. Parker, mcGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, new York; and Eliel, e. And Wilen, s., stereochemistry of Organic Compounds (1994) John Wiley & Sons, inc., new York. Many organic compounds exist in optically active form, i.e. they have the ability to rotate plane-polarized light planes. In describing optically active compounds, the prefixes D and L or R and S are used to represent the absolute configuration of the molecule about its chiral center. The prefixes d and l or (+) and (-) are used to denote the sign of the rotation of the compound to plane polarized light, where (-) or 1 indicates that the compound is left-handed. Compounds with (+) or d prefix are dextrorotatory. These stereoisomers are identical for a given chemical structure, except that they are mirror images of each other. A particular stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often referred to as an enantiomeric mixture. The 50:50 mixture of enantiomers is referred to as a racemic mixture or racemate, which may occur without stereoselectivity or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two optically inactive enantiomeric species.
Other terms, definitions and abbreviations herein include: wild type ("WT"); cysteine engineered mutant antibodies ("thio"); light chain ("LC"); heavy chain ("HC"); 6-maleimidocaproyl ("MC"); maleimidopropionyl ("MP"); valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyl ("PAB") and p-aminobenzyloxycarbonyl ("PABC"); a118C (EU numbering) of heavy chain=a121c (sequential numbering) =a169c (Kabat numbering) of light chain, K149C (Kabat numbering). Other definitions and abbreviations are provided herein.
II chemical degradation inducer
Chemical degradation inducer (CIDE) molecules can be conjugated to antibodies to form "Ab-CIDE" conjugates. The antibodies are conjugated via a linker (L1) to a CIDE ("D"), wherein the CIDE comprises a ubiquitin E3 ligase binding group ("E3 LB"), a linker ("L2") and a protein binding group ("PB"). Ab-CIDE molecule has the general formula:
Ab―(L1―D) p ，
wherein D is CIDE having the structure E3 LB-L2-PB; wherein E3LB is an E3 ligase binding group covalently bound to L2. L2 is a linker covalently bound to E3LB and PB; PB is a protein binding group covalently bound to L2. Ab is an antibody that covalently binds to L1; l1 is a linker covalently bound to Ab and D; p has a value of about 1 to about 50. The variable p reflects that the antibody may be linked to one or more L1-D groups. In one embodiment, p is about 1 to 8. In another embodiment, p is about 2.
The following sections describe the components that make up Ab-CIDE. In order to obtain Ab-CIDE with effective efficacy and desired therapeutic index, the following components are provided.
1. Antibody (Ab)
As described herein, antibodies, such as monoclonal antibodies (mAB), are used to deliver CIDE to target cells, such as cells expressing a particular protein targeted by the antibody. The antibody portion of the Ab-CIDE may target antigen expressing cells, thereby delivering antigen specific Ab-CIDE cells to the target cells, typically by endocytosis. Although Ab-CIDE comprising antibodies to antigens not found on the cell surface may result in reduced specific intracellular delivery of the CIDE moiety into the cell, the Ab-CIDE may still undergo pinocytosis. The Ab-CIDE and methods of using the same described herein advantageously utilize antibodies to cell surface recognition and/or endocytosis of the Ab-CIDE to deliver CIDE moieties into cells.
In particular embodiments, the antibody is thiomab, as described in detail below. Thiomab may have a modulated Fc effector, such as a LALAPG or NG2LH mutation. Furthermore, combinations are contemplated such that any antibody target (CD 71, trop2, MSLN, naPi2b, ly6E, epCAM, and CD 22) can be combined with any suitable combination of a thiomab mutation with any Fc effector modulation (including LALAPG or NG2LH mutation).
a. Human antibodies
In certain embodiments, the antibodies provided herein are human antibodies. Various techniques known in the art may be used to produce human antibodies. For a general description of human antibodies, see: van Dijk and van de Winkel, curr. Opin. Pharmacol.5:368-74 (2001) and Lonberg, curr. Opin. Immunol.20:450-459 (2008).
Human antibodies can be prepared by: the immunogen is administered to a transgenic animal that has been modified to produce a fully human antibody or a fully antibody having a human variable region in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci that replace endogenous immunoglobulin loci, either present extrachromosomal to the animal or randomly integrated into the animal's chromosome. In such transgenic mice, the endogenous immunoglobulin loci have typically been inactivated. For a review of methods of obtaining human antibodies from transgenic animals, see Lonberg, nat. Biotech.23:1117-1125 (2005). See also, e.g., descriptions xenomouise TM Technical U.S. Pat. nos. 6,075,181 and 6,150,584; description of the invention
Figure BDA0004113777320000221
Technical U.S. patent No. 5,770,429; description of K-M- >
Figure BDA0004113777320000222
Technical U.S. Pat. No. 7,041,870 and description->
Figure BDA0004113777320000223
Technical U.S. patent application publication No. US 2007/0061900). Human variable regions from whole antibodies produced by such animals may be further modified, for example, by combining with different human constant regions.
Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines for the production of human monoclonal antibodies have been described. (see, e.g., kozbor J.Immunol.,133:3001 (1984); brodeur et al, monoclonal Antibody Production Techniques and Applications, pages 51-63 (Marcel Dekker, inc., new York, 1987); and Boerner et al, J.Immunol.,147:86 (1991)). Human antibodies produced by human B cell hybridoma technology can also be described as follows: li et al, proc.Natl.Acad.Sci.USA,103:3557-3562 (2006). Additional methods include, for example, those described in U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, xiandai Mianyixue,26 (4): 265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, histology and Histopathology,20 (3): 927-937 (2005) and Vollmers and Brandlein, methods and Findings in Experimental and Clinical Pharmacology,27 (3): 185-91 (2005).
Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from a human phage display library. Such variable domain sequences can then be combined with the intended human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.
b. Antibodies derived from libraries
Antibodies for Ab-CIDE can be isolated by screening combinatorial libraries for antibodies having one or more desired activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries to obtain antibodies with desired binding characteristics. For reviews of such methods see, for example, hoogenboom et al, incorporated by reference in Methods in Molecular Biology 178:178:1-37 (O' Brien et al, human Press, totowa, NJ, 2001), and for further description see, for example, mcCafferty et al, nature 348:552-554; clackson et al, nature 352:624-628 (1991); marks et al, J.mol.biol.222:581-597 (1992); marks and Bradbury, incorporated by reference Methods in Molecular Biology 248:161-175 (Lo Main, human Press, totowa, N.J., 2003); sidhu et al, J.mol.biol.338 (2): 299-310 (2004); lee et al, J.mol.biol.340 (5): 1073-1093 (2004); felloose, proc. Natl. Acad. Sci. USA 101 (34): 12467-12472 (2004); and Lee et al, J.Immunol. Methods 284 (1-2): 119-132 (2004).
In some phage display methods, all components of the VH and VL genes are cloned individually by Polymerase Chain Reaction (PCR) and randomly recombined in a phage library from which antigen-binding phage can then be screened as described in: winter et al, ann.Rev.Immunol.,12:433-455 (1994). Phage typically display antibody fragments as single chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, the initial repertoire (e.g., from humans) can be cloned to provide a single source of antibodies to a wide range of non-self and self-antigens without any immunization, as described in: griffiths et al, EMBO J,12:725-734 (1993). Finally, the initial library can also be synthesized by: cloning unrearranged V gene segments from stem cells; and encoding highly variable CDR3 regions and accomplishing in vitro rearrangement using PCR primers containing random sequences, as described in: hoogenboom and Winter, J.mol.biol.,227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: us patent No. 5,750,373, and us publication nos. 2005/007974, 2005/019455, 2005/0266000, 2007/017126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.
Antibodies or antibody fragments isolated from a human antibody library are herein considered human antibodies or human antibody fragments.
c. Chimeric and humanized antibodies
In certain embodiments, the antibodies provided herein are chimeric antibodies. Some chimeric antibodies are described in the following documents: for example, U.S. Pat. No. 4,816,567 and Morrison et al, proc.Natl. Acad.Sci.USA,81:6851-6855 (1984). In one example, the chimeric antibody includes a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate (such as a monkey)) and a human constant region. In another example, a chimeric antibody is a "class switch" antibody in which the class or subclass has been altered from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
In certain embodiments, the chimeric antibody is a humanized antibody. Typically, the non-human antibodies are humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. In general, a humanized antibody comprises one or more variable domains in which the HVRs (e.g., CDRs (or portions thereof)) are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence.
Humanized antibodies and methods for their preparation are reviewed as described in: for example, almagro and Franson, front. Biosci.13:1619-1633 (2008), as described in the following: for example, riechmann et al Nature 332:323-329 (1988); queen et al, proc.Nat' lAcad.Sci.USA 86:10029-10033 (1989); U.S. Pat. nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; kashmiri et al Methods 36:25-34 (2005) (describing SDR (a-CDR) porting); padlan, mol. Immunol.28:489-498 (1991) (description "surface remolding"); dall' Acqua et al, methods 36:43-60 (2005) (description "FR shuffling"); and Osbourn et al, methods 36:61-68 (2005) and Klimka et al, br.J.cancer,83:252-260 (2000) (describes the "guided selection" approach to FR shuffling).
Human framework regions useful for humanization include, but are not limited to: the framework regions were selected using the "best fit" method (see, e.g., sims et al J. Immunol.151:2296 (1993)); framework regions of consensus sequences of human antibodies derived from specific subsets of light or heavy chain variable regions (see, e.g., carter et al Proc. Natl. Acad. Sci. USA,89:4285 (1992); and Presta et al J. Immunol.,151:2623 (1993)); human mature (somatic mutation) framework regions or human germline framework regions (see, e.g., almagro and Fransson, front. Biosci.13:1619-1633 (2008)); and framework regions from screening FR libraries (see, e.g., baca et al, J. Biol. Chem.272:10678-10684 (1997) and Rosok et al, J. Biol. Chem.271:22611-22618 (1996)).
d. Multispecific antibodies
In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. As used herein, the term "multispecific antibody" refers to an antibody that comprises an antigen-binding domain that has a multispecific specificity (i.e., an antibody that is capable of binding to two or more different epitopes on one molecule, or an epitope on two or more different molecules).
In some embodiments, the multispecific antibody is a monoclonal antibody (e.g., bispecific antibody) having binding specificities for at least two different antigen combining sites. In some embodiments, the first antigen binding domain and the second antigen binding domain of a multispecific antibody may bind to two epitopes within one and the same molecule (intramolecular binding). For example, the first antigen binding domain and the second antigen binding domain of a multispecific antibody may bind to two different epitopes on the same protein molecule. In certain embodiments, two different epitopes bound by a multispecific antibody are epitopes that are not normally bound at the same time by a single specific antibody (e.g., a conventional antibody or an immunoglobulin single variable domain). In some embodiments, the first antigen binding domain and the second antigen binding domain of a multispecific antibody may bind to epitopes located within two different molecules (intermolecular binding). For example, a first antigen binding domain of a multispecific antibody may bind to one epitope on one protein molecule, while a second antigen binding domain of a multispecific antibody may bind to another epitope on a different protein molecule, thereby crosslinking the two molecules.
In some embodiments, the antigen-binding domain of a multispecific antibody (such as a bispecific antibody) comprises two VH/VL units, wherein a first VH/VL unit binds to a first epitope and a second VH/VL unit binds to a second epitope, wherein each VH/VL unit comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). Such multispecific antibodies include, but are not limited to, full-length antibodies, antibodies having two or more VL and VH domains, and antibody fragments (such as Fab, fv, dsFv, scFv, diabodies, bispecific diabodies, and triabodies, antibody fragments that have been covalently or noncovalently linked). VH/VL units further comprising at least a portion of a heavy chain variable region and/or at least a portion of a light chain variable region may also be referred to as "arms", "half bodies" or "half antibodies". In some embodiments, the half comprises a sufficient portion of the heavy chain variable region to effect intramolecular disulfide bond formation with the second half. In some embodiments, the half-body comprises a knob mutation (knob mutation) or a hole mutation (hole mutation), e.g., to effect heterodimerization with a second half-body or half-antibody comprising a complementary hole mutation or knob mutation. The pestle and socket mutations are discussed further below.
In certain embodiments, the multispecific antibodies provided herein can be bispecific antibodies. As used herein, the term "bispecific antibody" is a multispecific antibody comprising an antigen-binding domain that is capable of specifically binding to two different epitopes on one biomolecule or is capable of specifically binding to an epitope on two different biomolecules. Bispecific antibodies may also be referred to herein as having "dual specificity" or as being "dual specific. Exemplary bispecific antibodies can bind proteins and any other antigens. In certain embodiments, one of the binding specificities is for protein and the other is for CD 3. See, for example, U.S. Pat. No. 5,821,337. In certain embodiments, a bispecific antibody can bind to two different epitopes of the same protein molecule. In certain embodiments, bispecific antibodies can bind to two different epitopes on two different protein molecules. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing proteins. Bispecific antibodies can be made as full length antibodies or antibody fragments.
Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see, milstein and Cuello, nature 305:537 (1983), WO 93/08829, and Traunecker et al, EMBO J.10:3655 (1991)) and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168, WO2009/089004, U.S. Pat. No. 5,731,168,WO2009/089004, U.S. Pat. No. 5/0182127, U.S. Pat. No. 2011/0287009, marvin and Zhu, acta Pharmacol.sin (2005) 26 (6): 649-658, and Kontemann (2005) Acta Pharmacol.sin., 26:1-9). As used herein, the term "knob-in-hole" or "KnH" technique refers to a technique that directs the pairing of two polypeptides together in vivo or in vitro by introducing a protrusion (knob) into one polypeptide and a cavity (hole) into the other polypeptide at the interface where they interact. For example, knH has been introduced in the Fc: fc binding interface, CL: CH1 interface or VH/VL interface of antibodies (see, e.g., U.S. Pat. No. 5,2011/0287009; U.S. Pat. No. 5,2007/0178552; WO 96/027011; WO 98/050431; zhu et al, 1997,Protein Science 6:781-788; and WO 2012/106587). In some embodiments, knH drives two different heavy chains paired together during the preparation of the multispecific antibody. For example, a multispecific antibody having KnH in its Fc region may further comprise a single variable domain linked to the respective Fc region, or further comprise a different heavy chain variable domain paired with a similar or different light chain variable domain. The KnH technique can also be used to pair together two different receptor ectodomains or any other polypeptide sequences comprising different target recognition sequences (e.g., including affibodies, peptibodies, and other Fc fusions).
As used herein, the term "knob mutation" refers to a mutation that introduces a protrusion (knob) into a polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptides have a hole mutation.
As used herein, the term "socket mutation" refers to a mutation that introduces a cavity (socket) into a polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptides have a knob mutation.
"protuberance" refers to at least one amino acid side chain that protrudes from the interface of a first polypeptide and thus can be positioned in a compensation chamber of an adjacent interface (i.e., the interface of a second polypeptide) to stabilize a heteromultimer, e.g., to favor heteromultimer formation over homomultimer formation. The protrusions may be present in the original interface or may be synthetically introduced (e.g., by altering the nucleic acid encoding the interface). In some embodiments, the nucleic acid encoding the interface of the first polypeptide is altered to encode a protuberance. To this end, the nucleic acid encoding at least one "original" amino acid residue in the interface of the first polypeptide is replaced with a nucleic acid encoding at least one "input" amino acid residue (which has a larger side chain volume than the original amino acid residue). It will be appreciated that there may be more than one original and corresponding input residue. The side chain volumes of the various amino residues are shown, for example, in table 1 of US 2011/0287009. The mutation that introduces the "protrusion" may be referred to as a "pestle mutation".
In some embodiments, the input residue for forming the protuberance is a naturally occurring amino acid residue selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). In some embodiments, the input residue is tryptophan or tyrosine. In some embodiments, the original residues used to form the protrusions have a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.
By "cavity" is meant at least one amino acid side chain recessed from the interface of the second polypeptide, thus accommodating a corresponding protrusion on the adjacent interface of the first polypeptide. The cavity may be present in the original interface, or may be synthetically introduced (e.g., by altering the nucleic acid encoding the interface). In some embodiments, the nucleic acid encoding the interface of the second polypeptide is altered to encode a cavity. To this end, the nucleic acid encoding the at least one "original" amino acid residue in the interface of the second polypeptide is replaced with a DNA encoding the at least one "input" amino acid residue (which has a smaller side chain volume than the original amino acid residue). It will be appreciated that there may be more than one original and corresponding input residue. In some embodiments, the input residues used to form the cavity are naturally occurring amino acid residues selected from the group consisting of alanine (a), serine (S), threonine (T), and valine (V). In some embodiments, the input residue is serine, alanine, or threonine. In some embodiments, the original residues used to form the cavity have a large side chain volume, such as tyrosine, arginine, phenylalanine, or tryptophan. The abrupt change introduced into the "cavity" may be referred to as a "acetabular abrupt change".
The protrusion is "locatable" in the cavity, which means that the spatial location of the protrusion and the cavity at the interface of the first polypeptide and the second polypeptide, respectively, and the size of the protrusion and the cavity are such that the protrusion can be located in the cavity without significantly interfering with the normal association of the first polypeptide and the second polypeptide at the interface. Since protrusions (such as Tyr, phe, and Trp) generally do not extend perpendicularly from the axis of the interface, but rather have a preferred conformation, in some instances, the alignment of the protrusions with the corresponding cavities may depend on the method of modeling the protrusion/cavity pairs based on: three-dimensional structures such as those obtained by X-ray crystallography or Nuclear Magnetic Resonance (NMR). This can be accomplished using techniques widely accepted in the art.
In some embodiments, the knob mutation in the IgG1 constant region is T366W (EU numbering). In some embodiments, the mortar mutation in the IgG1 constant region comprises one or more mutations selected from T366S, L368A and Y407V (EU numbering). In some embodiments, the mortar mutations in the IgG1 constant region comprise T366S, L368A and Y407V (EU numbering).
In some embodiments, the knob mutation in the IgG4 constant region is T366W (EU numbering). In some embodiments, the mortar mutation in the IgG4 constant region comprises one or more mutations selected from T366S, L368A and Y407V (EU numbering). In some embodiments, the mortar mutation in the IgG4 constant region comprises T366S, L368A and Y407V (EU numbering).
Multispecific antibodies can also be prepared by the following method: engineering electrostatic control effects to produce antibody Fc-heterodimer molecules (WO 2009/089004 A1); crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al, science,229:81 (1985)), producing bispecific antibodies using leucine zippers (see, e.g., kostelny et al, J.Immunol.,148 (5): 1547-1553 (1992)), preparing bispecific antibody fragments using "diabody" techniques (see, e.g., hollinger et al, proc. Natl. Acad. Sci. USA,90:6444-6448 (1993)), and preparing trispecific antibodies using single chain Fv (sFv) dimers (see, e.g., gruber et al, J.Immunol.,152:5368 (1994)), and following methods such as described in Tutt et al, J.Immunol.147:60 (1991).
Also included herein are engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies" or "dual variable domain immunoglobulins" (DVD) (see, e.g., US 2006/0025576A1; and Wu et al Nature Biotechnology (2007)). Antibodies or fragments herein also include "dual-acting FAb" or "DAF" which comprise antigen binding sites that bind to a target protein and a different antigen (see, e.g., US 2008/0069820).
e. Antibody fragments
In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, fab '-SH, F (ab') 2 Fv, and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson et al, nat.Med.9:129-134 (2003). For reviews of scFv fragments, see, e.g., plucktHun, the Pharmacology of Monoclonal Antibodies, vol.113, rosenburg and Moore, inc. (Springer-Verlag, new York), pages 269-315 (1994); see also WO 93/16185; and U.S. patent nos. 5,571,894 and 5,587,458. For Fab fragments and F (ab') comprising salvage receptor binding epitope residues and having an extended in vivo half-life 2 See U.S. Pat. No. 5,869,046 for discussion of fragments.
Diabodies are antibody fragments having two antigen binding sites, which may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; hudson et al, nat.Med.9:129-134 (2003); and Hollinger et al, proc.Natl. Acad. Sci. USA 90:6444-6448 (1993). For a description of trisomy and tetrasomy antibodies see also Hudson et al, nat. Med.9:129-134 (2003).
A single domain antibody is an antibody fragment comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (domntis, inc., waltham, MA; see, e.g., U.S. patent No. 6,248,516B1).
Antibody fragments can be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E.coli or phage), as described herein.
f. Antibody variants
In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of antibodies. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequence of an antibody. Any combination of deletions, insertions, and substitutions can be made to achieve the final construct, provided that the final construct has the desired characteristics, e.g., antigen binding.
g. Recombinant methods and compositions
Recombinant methods and compositions can be used to produce antibodies, for example, as described in U.S. Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an antibody described herein is provided. Such nucleic acids may encode amino acid sequences comprising the VL of an antibody and/or amino acid sequences comprising the VH of an antibody (e.g., the light chain and/or heavy chain of an antibody). In further embodiments, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In further embodiments, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises the following (e.g., has been transformed with): (1) A vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of an antibody and an amino acid sequence comprising a VH of an antibody; or (2) a first vector comprising a nucleic acid encoding an amino acid sequence of a VL of an antibody and a second vector comprising a nucleic acid encoding an amino acid sequence of a VH of an antibody. In one embodiment, the host cell is a eukaryotic cell, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, sp20 cell). In one embodiment, a method of producing an antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
For recombinant production of antibodies, nucleic acids encoding the antibodies (e.g., as described above) are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of an antibody).
Suitable host cells for cloning or expressing the antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. nos. 5,648,237, 5,789,199, and 5,840,523. (see also Charlton, methods in Molecular Biology, volume 248 (B.K.C.Lo, B.K.C., main edition, humana Press, totowa, NJ (2003), pages 245-254, describing the expression of antibody fragments in E.coli.) antibodies can be isolated from bacterial cell pastes in soluble fractions after expression and can be further purified.
In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast, including fungal and yeast strains whose glycosylation pathways have been "humanized" such that antibodies with a partially or fully human glycosylation pattern are produced, are also suitable cloning or expression hosts for vectors encoding antibodies. See Gerngross, nat. Biotech.22:1409-1414 (2004); and Li et al, nat.Biotech.24:210-215 (2006).
Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified that can be used with insect cells, particularly for transfection of Spodoptera frugiperda (Spodoptera frugiperda) cells.
Plant cell cultures may also be used as hosts. See, e.g., U.S. Pat. nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 (describing PLANTIBODIES for antibody production in transgenic plants) TM Technology).
Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines include: monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (e.g., graham et al, J. Gen. Virol.36:59 (1977) 293 or 293 cells, baby hamster kidney cells (BHK), mouse support cells (e.g., TM4 cells as described in Mather, biol. Reprod.23:243-251 (1980)), monkey kidney cells (CV 1), african green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumors (MMT 060562)), TRI cells (e.g., as described in Mather et al, annals N. Y. Acad. Sci.383:44-68 (1982)), MRC 5 cells, and FS4 other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR - CHO cells (Urlaub et al, proc.Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., yazaki and Wu, methods in Molecular Biology, volume 248 (b.k.c.lo master, humana Press, totowa, NJ), pages 255-268 (2003).
Referring now to antibody affinity, in embodiments, the antibody binds to one or more tumor-associated antigens or cell surface receptors. In embodiments, the tumor-associated antigen or cell surface receptor is selected from the group consisting of CD71, trop2, MSLN, naPi2b, ly6E, epCAM, and CD22.
As described herein, ab-CIDE may comprise an antibody, for example an antibody selected from the group consisting of:
i. anti-Ly 6E antibodies
Ly6E (lymphocyte antigen 6 complex gene locus E; ly67, RIG-E, SCA-2, TSA-1); np_002337.1; NM_002346.2; de Nooij-van Dalen, A.G. et al (2003) int.J. cancer 103 (6), 768-774; zammit, D.J. et al (2002) mol.cell.biol.22 (3): 946-952; WO 2013/17705.
In certain embodiments, the Ab-CIDE may comprise an anti-Ly 6E antibody. Lymphocyte antigen 6 complex locus E (Ly 6E), also known as retinoic acid-inducible gene E (RIG-E) and stem cell antigen 2 (SCA-2). It is a GPI-linked, 131 amino acid long, approximately 8.4kDa protein with unknown function and no known binding partner. It was originally identified as a transcript expressed in immature thymocytes, thymic medullary epithelial cells in mice (Mao et al (1996) proc.Natl. Acad. Sci.U.S. A.93:5910-5914). In some embodiments, the subject matter described herein provides an Ab-CIDE comprising an anti-Ly 6E antibody described in PCT publication No. WO 2013/177055.
In some embodiments, the subject matter described herein provides Ab-CIDE comprising an anti-Ly 6E antibody comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID No. 4; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 5; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 6; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 1; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 2; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 3.
In one aspect, the subject matter described herein provides an Ab-CIDE comprising an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID No. 4; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 5; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 6. In another embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID No. 4; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 5; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 6.
In another aspect, the subject matter described herein provides an Ab-CIDE comprising an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 1; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 2; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 3. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 1; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 2; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 3.
In another aspect, the Ab-CIDE comprises an antibody comprising: (a) A VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 4; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 5; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 6; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 1, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 2, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 3.
In another aspect, the subject matter described herein provides an Ab-CIDE comprising an antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID No. 4; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 5; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 6; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 1; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 2; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 3.
In any of the above embodiments, the anti-Ly 6E antibody of Ab-CIDE is humanized. In one embodiment, the anti-Ly 6E antibody comprises the HVR of any one of the embodiments described above, and further comprises a human acceptor framework, e.g., a human immunoglobulin framework or a human consensus framework.
In another aspect, an anti-Ly 6E antibody to Ab-CIDE comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID No. 8. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID No. 8 comprises a substitution (e.g., a conservative substitution), insertion or deletion relative to a reference sequence, but an anti-Ly 6E antibody comprising the sequence retains the ability to bind Ly 6E. In certain embodiments, in SEQ ID NO. 8, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In certain embodiments, in SEQ ID NO. 8, a total of 1 to 5 amino acids are substituted, inserted and/or deleted. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVR (i.e., in the FR). Optionally, the anti-Ly 6E antibody comprises the VH sequence of SEQ ID NO. 8, including post-translational modifications of that sequence. In a specific embodiment, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 4, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 5, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 6.
In another aspect, an anti-Ly 6E antibody of Ab-CIDE is provided, wherein the antibody comprises a light chain variable domain (Vl) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO. 7. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 7 comprises a substitution (e.g., a conservative substitution), insertion or deletion relative to a reference sequence, but an anti-Ly 6E antibody comprising the sequence retains the ability to bind Ly 6E. In certain embodiments, in SEQ ID NO. 7, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In certain embodiments, in SEQ ID NO. 7, a total of 1 to 5 amino acids are substituted, inserted and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the anti-Ly 6E antibody comprises the VL sequence of SEQ ID NO. 7, including post-translational modifications of that sequence. In a specific embodiment, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 1; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 2; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 3.
In another aspect, an Ab-CIDE comprising an anti-Ly 6E antibody is provided, wherein the antibody comprises a VH in any of the embodiments as provided above and a VL in any of the embodiments as provided above.
In one embodiment, an Ab-CIDE is provided wherein the antibody comprises the VH and VL sequences of SEQ ID NO. 8 and SEQ ID NO. 7, respectively, including post-translational modifications of those sequences.
In another aspect, provided herein is an Ab-CIDE comprising an antibody that binds to the same epitope as an anti-Ly 6E antibody provided herein. For example, in certain embodiments, an Ab-CIDE is provided that comprises an antibody that binds to the same epitope as an anti-Ly 6E antibody (comprising the VH sequence of SEQ ID NO:8 and the VL sequence of SEQ ID NO:7, respectively).
In another aspect, an anti-Ly 6E antibody of Ab-CIDE according to any of the above embodiments is a monoclonal antibody, including a human antibody. In one embodiment, the anti-Ly 6E antibody of Ab-CIDE is an antibody fragment, e.g., fv, fab, fab ', scFv, diabody or F (Ab') 2 Fragments. In another embodiment, the antibody is a substantially full length antibody, such as an IgG1 antibody, an IgG2a antibody, or other antibody types or isotypes as defined herein. In some embodiments, ab-CIDE comprises an anti-Ly 6E antibody comprising a heavy chain and a light chain comprising the amino acid sequences of SEQ ID NO. 10 and SEQ ID NO. 9, respectively.
anti-NaPi 2b antibodies
Napi2b (Napi 3b, NAPI-3B, NPTIIb, SLC A2, solute carrier family 34 (sodium phosphate), member 2, sodium-dependent phosphate transporter type II 3b, genbank accession NM-006424) J.biol.chem.277 (22): 19665-19672 (2002), genomics 62 (2): 281-284 (1999); feild, J.A. et al (1999) biochem. Biophys. Res. Commun.258 (3): 578-582); WO2004022778 (claim 2); EP1394274 (example 11); WO2002102235 (claim 13; page 326); EP875569 (claim 1; pages 17 to 19); WO200157188 (claim 20; page 329); WO2004032842 (example IV); WO200175177 (claim 24; pages 139 to 140); cross-reference: 604217 as MIM; np_006415.1; NM_006424_1.
In certain embodiments, the Ab-CIDE comprises an anti-NaPi 2b antibody.
In some embodiments, described herein is an Ab-CIDE comprising an anti-NaPi 2b antibody comprising at least one, two, three, four, five, or six HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 12; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 13; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 14; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 15; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 16.
In one aspect, described herein is an Ab-CIDE comprising an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 12; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 13. In another embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 12; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 13.
In another aspect, described herein is an Ab-CIDE comprising an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 14; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 15; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 16. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 14; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 15; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 16.
In another aspect, the Ab-CIDE comprises an antibody comprising: (a) A VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 12; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 13; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 14, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 15, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 16.
In another aspect, described herein is an Ab-CIDE comprising an antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 12; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 13; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 14; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 15; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 16.
In any of the above embodiments, the anti-NaPi 2b antibody of Ab-CIDE is humanized. In one embodiment, the anti-NaPi 2b antibody comprises the HVR of any one of the embodiments described above, and further comprises a human acceptor framework, such as a human immunoglobulin framework or a human consensus framework.
In another aspect, an anti-NaPi 2b antibody of Ab-CIDE comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID No. 17. In certain embodiments, a VH sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 54 comprises a substitution (e.g., a conservative substitution), insertion or deletion relative to a reference sequence, but an anti-NaPi 2b antibody comprising that sequence retains the ability to bind to NaPi2 b. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO. 17. In certain embodiments, a total of 1 to 5 amino acids are substituted, inserted and/or deleted in SEQ ID NO. 17. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVR (i.e., in the FR). Optionally, the anti-NaPi 2b antibody comprises the VH sequence of SEQ ID NO. 17, including post-translational modifications of that sequence. In a specific embodiment, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 11, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 12, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 13.
In another aspect, an anti-NaPi 2b antibody to Ab-CIDE is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO. 18. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 18 comprises a substitution (e.g., a conservative substitution), insertion or deletion relative to a reference sequence, but an anti-NaPi 2b antibody comprising that sequence retains the ability to bind to anti-NaPi 2 b. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO. 18. In certain embodiments, a total of 1 to 5 amino acids are substituted, inserted and/or deleted in SEQ ID NO. 18. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the anti-NaPi 2b antibody comprises the VL sequence of SEQ ID NO. 18, including post-translational modifications of that sequence. In a specific embodiment, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 14; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 15; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 16.
In another aspect, an Ab-CIDE comprising an anti-NaPi 2b antibody is provided, wherein the antibody comprises a VH in any of the embodiments as provided above and a VL in any of the embodiments as provided above.
In one embodiment, an Ab-CIDE is provided wherein the antibody comprises the VH and VL sequences of SEQ ID NO. 17 and SEQ ID NO. 18, respectively, including post-translational modifications of those sequences.
In another aspect, provided herein is an Ab-CIDE comprising an antibody that binds to the same epitope as an anti-NaPi 2b antibody provided herein. For example, in certain embodiments, an Ab-CIDE is provided that comprises an antibody that binds to the same epitope as an anti-NaPi 2b antibody (comprising the VH sequence of SEQ ID NO:17 and the VL sequence of SEQ ID NO:18, respectively).
In another aspect, an anti-NaPi 2b antibody of an Ab-CIDE according to any of the embodiments above is a monoclonal antibody, including a human antibody. In one embodiment, the anti-NaPi 2b antibody of Ab-CIDE is an antibody fragment, e.g., fv, fab, fab ', scFv, diabody or F (Ab') 2 Fragments. In another embodiment, the antibody is a substantially full length antibody, such as an IgG1 antibody, an IgG2a antibody, or other antibody types or isotypes as defined herein.
anti-CD 22 antibodies
CD22 (B cell receptor CD22-B isoform, BL-CAM, lyb-8, lyb8, SIGLEC-2, FLJ22814, genbank accession No. AK 026467); wilson et al (1991) J.Exp. Med.173:137-146; WO2003072036 (claim 1; fig. 1); cross-reference: 107266 as MIM; np_001762.1; NM_001771_1.
In certain embodiments, ab-CIDE may comprise an anti-CD 22 antibody comprising three light chain hypervariable regions (HVR-L1, HVR-L2, and HVR-L3) and three heavy chain hypervariable regions (HVR-H1, HVR-H2, and HVR-H3). In one embodiment, the anti-CD 22 antibody of Ab-CIDE comprises three light chain hypervariable regions and three heavy chain hypervariable regions (SEQ ID NOS: 19-24), the sequences of which are shown below. In one embodiment, the anti-CD 22 antibody of Ab-CIDE comprises the variable light chain sequence of SEQ ID NO. 25 and the variable heavy chain sequence of SEQ ID NO. 26. In one embodiment, the anti-CD 22 antibody of Ab-CIDE of the invention comprises the light chain sequence of SEQ ID NO. 27 and the heavy chain sequence of SEQ ID NO. 28.
anti-CD 71 antibodies
In certain embodiments, the Ab-CIDE may comprise an anti-CD 71 antibody. CD71 (transferrin receptor) is an intact membrane glycoprotein and plays an important role in the uptake of iron by cells. It is well known as a marker of cell proliferation and activation. Although all proliferating cells in the hematopoietic system express CD71, CD71 has been considered a useful erythrocyte-associated antigen. In any of the above embodiments, the anti-CD 71 antibody of Ab-CIDE is humanized.
In one embodiment, the anti-CD 71 antibody comprises a NG2LH modification that is a combination of the N297G mutation and the IgG2 lower hinge region that reduces/eliminates IgG1 mAb effector function. In another embodiment, the anti-CD 71 antibody comprises an engineered Cys residue for binding to the linker. In one embodiment, a description of the parent IgG1 mAb lacking all of these changes is found in: WO2016081643, which is incorporated herein by reference in its entirety.
In an embodiment, the anti-CD 71 antibody is anti-hutfr1.higg1.lc.k1496.hc.l174c.y 373c.ng2lh ABP1AA25970 (high affinity DAR 6). In one embodiment, the anti-CD 71 antibody of Ab-CIDE comprises: the light chain sequence of SEQ ID NO. 30 and the heavy chain sequence of SEQ ID NO. 29.
In an embodiment, the anti-CD 71 antibody is anti-hutfr2.higgg1.lc.k1496.hc.l174c.y 373c.ng2lh ABP1AA25969 (low affinity DAR 6). In one embodiment, the anti-CD 71 antibody of Ab-CIDE comprises: light chain sequence of SEQ ID No. 32 = and heavy chain sequence of SEQ ID No. 31.
In an embodiment, the anti-CD 71 antibody is anti-hutfr1.higg1.lc.k1496.ng 2lh ABP1AA30139 (high affinity DAR 2). In one embodiment, the anti-CD 71 antibody of Ab-CIDE comprises: the light chain sequence of SEQ ID NO. 34 and the heavy chain sequence of SEQ ID NO. 33.
In an embodiment, the anti-CD 71 antibody is anti-hutfr2.higgg1.lc.k1496.ng 2lh ABP1AA30140 (low affinity DAR 2). In one embodiment, the anti-CD 71 antibody of Ab-CIDE comprises: the light chain sequence of SEQ ID NO. 36 and the heavy chain sequence of SEQ ID NO. 35.
v. anti-Trop 2 antibodies
In certain embodiments, the Ab-CIDE may comprise an anti-Trop 2 antibody. Trop2 (trophoblast antigen 2) is a transmembrane glycoprotein, an intracellular calcium signaling protein that has differential expression in a variety of cancers. It signals cells to self-renew, proliferate, invade and survive. Trop2 is also known as cell surface glycoprotein Trop-2/Trop2, gastrointestinal tumor associated antigen GA7331, pancreatic cancer marker protein GA733-1/GA733, membrane component chromosome 1 surface marker 1M1S1, epithelial glycoprotein-1, EGP-1, CAA1, and adhesive drip cornea dystrophy GDLD and TTD2. In any of the above embodiments, the anti-Trop 2 antibody of Ab-CIDE is humanized. In one embodiment, the anti-Trop 2 antibodies are described in US-2014/0377287 and US-2015/0366988, each of which is incorporated herein by reference in its entirety.
anti-MSLN antibodies
In certain embodiments, the Ab-CIDE may comprise an anti-MSLN antibody. MSLN (mesothelin) is a glycosyl phosphatidylinositol anchored cell surface protein that can be used as a cell adhesion protein. MSLNs are also known as CAK1 and MPF. This protein is overexpressed in epithelial mesothelioma, ovarian cancer and specific squamous cell carcinoma. In any of the above embodiments, the anti-MSLN antibody of Ab-CIDE is humanized. In one embodiment, the anti-MSLN antibody is h7d9.v3, for a description see Scales, s.j. Et al, mol.cancer ter.2014, 13 (11), 2630-2640, incorporated herein by reference in its entirety.
anti-EpCAM antibodies
In certain embodiments, the Ab-CIDE may comprise an anti-EpCAM antibody. In one aspect, the antibody to Ab-CIDE can be an antibody to a protein found on a variety of cell or tissue types. Examples of such antibodies include EpCAM. An epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that mediates ca2+ -independent homotypic intercellular adhesion in epithelial cells (Litvinov, s. Et al (1994) Journal of Cell Biology (2): 437-46). Also known as DIAR5, EGP-2, EGP314, EGP40, ESA, HNPCC8, KS1/4, KSA, M4S1, MIC18, MK-1, TACSTD1, TROP1, epCAM is also involved in cell signaling (Maetzel, D. Et al, (2009) Nature Cell Biology (2): 162-71), migration (Osta, WA; et al, (2004) Cancer Res.64 (16): 5818-24), proliferation and differentiation (Litvinov, S. Et al (1996) Am JPathol.148 (3): 865-75). In addition, epCAM has oncogenic potential by up-regulating the capacity of c-myc, E-fabp and cyclin A and E (Munz, M.et al (2004) Oncogene23 (34): 5748-58). EpCAM can be used as a diagnostic marker for various cancers, since EpCAM is only expressed in epithelial and epithelial-derived neoplasms. In other words, ab-CIDE can be used to deliver CIDE to a number of cells or tissues, rather than to a particular cell type or tissue type as is the case when using a targeting antibody.
1. Affinity for antibodies
In certain embodiments, the antibody provided herein has a dissociation constant (Kd) of ∈1 μM, +.100 nM, +.50 nM, +.10 nM, +.5 nM, +.1 nM, +.0.1 nM, +.0.01 nM or+.0.001 nM, and optionally +.10 nM -13 M (e.g. 10 -8 M or less, e.g. 10 -8 M to 10 -13 M, e.g. 10 -9 M to 10 -13 M)。
In one embodiment, kd is measured by radiolabeled antigen binding assay (RIA) with the antibody of interest and its antigen in Fab form, as described in the assay below. The solution binding affinity of Fab to antigen was achieved by using a minimum concentration in the presence of a series of unlabeled antigen titration 125 I) The labeled antigen balances the Fab and then the bound antigen is captured by an anti-Fab antibody coated plate (see, e.g., chen et al, j. Mol. Biol.293:865-881 (1999)). To determine the conditions for the assay, the assay was run with a 50mM sodium carbonate solution (pH 9.6) containing 5. Mu.g/ml of the capture anti-Fab antibody (Cappel Labs)
Figure BDA0004113777320000401
The multiwell plate (Thermo Scientific) was coated overnight and then blocked with a PBS solution containing 2% (w/v) bovine serum albumin for two to five hours at room temperature (about 23 ℃). In the non-adsorbed plate (Nunc# 269620), 100pM or 26pM [ 125 I]Antigen in admixture with serial dilutions of the target Fab (e.g., consistent with assessment of anti-VEGF antibody Fab-12 in Presta et al, cancer Res.57:4593-4599 (1997)). The target Fab was then incubated overnight The method comprises the steps of carrying out a first treatment on the surface of the However, incubation may last longer (e.g., about 65 hours) to ensure equilibrium is reached. Thereafter, the mixture was transferred to a capture plate for incubation at room temperature (e.g., one hour). The solution was then removed and 0.1% polysorbate 20 (Tween-/in PBS>
Figure BDA0004113777320000402
) The plates were washed eight times. When the plate has been dried, 150. Mu.l/well of scintillator (MICROSICINT-20 is added TM The method comprises the steps of carrying out a first treatment on the surface of the Packard), and at TOPCount TM The plates were counted for tens of minutes on a gamma counter (Packard). The concentration of each Fab that gave less than or equal to 20% of maximum binding was selected for use in the competitive binding assay.
According to another embodiment, the immobilized antigen CM5 chip is used at about 10 Response Units (RU) at 25℃
Figure BDA0004113777320000403
-2000 or->
Figure BDA0004113777320000404
-3000 (BIAcore, inc., piscataway, NJ), kd is measured by surface plasmon resonance assay. Briefly, carboxymethylated dextran biosensor chips (CM 5, BIACORE, inc.) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen was diluted to 5. Mu.g/ml (about 0.2. Mu.M) with 10mM sodium acetate pH 4.8, followed by injection at a flow rate of 5. Mu.l/min to obtain about 10 Response Units (RU) of conjugated protein. After antigen injection, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, injection was performed at 25℃at a flow rate of about 25. Mu.l/min at a temperature of about 0.05% polysorbate 20 (TWEEN 20 TM ) Two-fold serial dilutions (0.78 nM to 500 nM) of Fab in PBS of surfactant (PBST). Simple one-to-one Langmuir binding model was used (>
Figure BDA0004113777320000411
Evaluation Software 3.2 version 3.2) by simultaneous fitting of association and dissociation sensorsThe association rate (kon) and dissociation rate (koff) were calculated. The equilibrium dissociation constant (Kd) is calculated as the ratio koff/kon. See, for example, seyforth et al, J.mol. Biol.293:865-881 (1999). If the association rate exceeds 106M-1s-1 as determined by the above surface plasmon resonance measurement, the association rate can be determined by using a fluorescence quenching technique, i.e., as in a spectrometer such as a spectrophotometer (Aviv Instruments) or 8000 series SLM-AMINCO equipped with a flow stop device TM The increase or decrease in fluorescence emission intensity (excitation=295 nM; emission=340 nM,16nM bandpass) of 20nM anti-antigen antibody (Fab form) in PBS pH 7.2 at 25 ℃ was measured in a spectrophotometer (ThermoSpectronic) in the presence of increasing concentrations of antigen using a stirred cuvette.
2. Joint (L1)
As described herein, a "linker" (L1, linker 1) is a bifunctional or multifunctional moiety that can be used to attach one or more CIDE moieties (D) to an antibody (Ab) to form Ab-CIDE. In some embodiments, L1 having reactive functional groups for covalent attachment to CIDE and antibodies may be used to make Ab-CIDE. For example, in some embodiments, the cysteine thiol of an antibody (Ab) may form a bond with a reactive functional group of a linker or a linker L1-CIDE group to prepare Ab-CIDE. In particular, the chemical structure of the linker can have a significant impact on both the efficacy and safety of the Ab-CIDE (Ducry & Stump, bioconjugate Chem,2010,21,5-13). Selection of the correct linker will affect the correct delivery of the drug to the intended cellular compartment of the target cell.
In certain embodiments, the L1 linker may be a self-eliminating linker.
In certain embodiments, the L1 linker is selected from the group consisting of L1a, L1b, and L1 c:
examples of L1 a:
Figure BDA0004113777320000412
Figure BDA0004113777320000421
examples of L1 b:
Figure BDA0004113777320000422
examples of L1 c:
Figure BDA0004113777320000423
wherein ,
j is-CH 2 -CH 2 -CH 2 -NH-C(O)-NH 2 ；—CH 2 -CH 2 -CH 2 -CH 2 -
NH 2 ；—CH 2 -CH 2 -CH 2 -CH 2 -NH-CH 3 The method comprises the steps of carrying out a first treatment on the surface of the or-CH 2 -CH 2 -
CH 2 -CH 2 -N(CH 3 ) 2 ；
R 5 and R6 Independently hydrogen or C 1-5 An alkyl group; or R is 5 and R6 Together with the nitrogen to which each is attached, form an optionally substituted 5-to 7-membered heterocyclyl;
R 7 and R8 Each independently is hydrogen, halo, C 1-5 Alkyl, C 1-5 Alkoxy or hydroxy;
and wherein
Figure BDA0004113777320000431
Is the point of attachment to Ab.
In certain embodiments, the L1 linker is a hydrophilic self-eliminating linker. Examples of these types of L1 linkers are described in WO2014/100762, which is incorporated herein by reference in its entirety. L1 linkers include, but are not limited to, formulas I-XII.
The present disclosure provides an L1 linker of formula (I):
Figure BDA0004113777320000432
or a salt, solvate or stereoisomer thereof;
wherein ：
d is a drug moiety or CIDE;
t is a targeting moiety, such as an antibody;
x is a hydrophilic self-eliminating linker;
L 1 is different from L1 and is a bond, a second self-eliminating linker or a cyclized self-eliminating linker;
L 2 is a bond or a second self-eliminating linker;
wherein if L 1 For a second self-eliminating linker or cyclized self-eliminating linker, then L is a bond;
Wherein if L 2 For the second self-eliminating joint, L 1 Is a bond;
L 3 is a peptide linker;
L 4 is a bond or a spacer; and
a is an acyl unit.
In some embodiments, an L1 linker of formula (la) is provided:
Figure BDA0004113777320000433
or a salt or solvate or stereoisomer thereof; therein D, T, X, L 1 、L 2 、L 3 、L 4 And A is as defined for formula (I) and p is 1 to 20. In some embodiments, p is 1 to 8. In some embodiments, p is 1 to 6. In some embodiments, p is 1 to 4. In some embodiments, p is 2 to 4. In some embodiments, p is 1, 2, 3, or 4.
The present disclosure also provides an L1 linker of formula (II):
Figure BDA0004113777320000441
or a salt, solvate or stereoisomer thereof;
wherein ：
d is a drug moiety or CIDE;
t is a targeting moiety or antibody;
R 1 is hydrogen, unsubstituted or substituted C 1-3 Alkyl, or unsubstituted or substituted heterocyclyl;
L 1 is a bond, a second self-eliminating linker or a cyclized self-eliminating linker; 2
L 2 Is a bond, a second self-eliminating linker;
wherein if L 1 For a second self-eliminating linker or cyclized self-eliminating linker, L 2 Is a bond;
wherein if L 1 For the second self-eliminating joint, L 2 Is a bond;
L 3 is a peptide linker;
L 4 is a bond or a spacer; and
a is an acyl unit.
In some embodiments, an L1 linker of formula (IIa) is provided:
Figure BDA0004113777320000442
Or a salt or solvate or stereoisomer thereof; therein D, T, L 1 、L 2 、L 3 、L 4 And A is as defined for formula (II) and p is 1 to 20. In some embodiments, p is 1 to 8. In some embodiments, p is 1 to 6. In some embodiments, p is 1 to 4. In some embodiments, p is 2 to 4. In some embodiments, p is 1, 2, 3, or 4.
The present disclosure also provides an L1 linker of formula (III):
Figure BDA0004113777320000451
or a salt, solvate or stereoisomer thereof;
wherein T is a targeting moiety.
In some embodiments, L1 linkers of formula (IIIa) are provided:
Figure BDA0004113777320000452
or a salt or solvate or stereoisomer thereof; wherein T is a targeting moiety and p is 1 to 20. In some embodiments, p is 1 to 8. In some embodiments, p is 1 to 6. In some embodiments, p is 1 to 4. In some embodiments, p is 2 to 4. In some embodiments, p is 1, 2, 3, or 4.
The present disclosure provides an L1 linker of formula (IV):
Figure BDA0004113777320000453
or a salt, solvate or stereoisomer thereof;
wherein T is a targeting moiety.
In some embodiments, L1 linkers of formula (IVa) are provided:
Figure BDA0004113777320000454
or a salt or solvate or stereoisomer thereof; wherein T is a targeting moiety and p is 1 to 20. In some embodiments, p is 1 to 8. In some embodiments, p is 1 to 6. In some embodiments, p is 1 to 4. In some embodiments, p is 2 to 4. In some embodiments, p is 1, 2, 3, or 4.
The present disclosure provides an L1 linker of formula (V):
Figure BDA0004113777320000461
or a salt, solvate or stereoisomer thereof;
wherein T is a targeting moiety.
In some embodiments, an L1 linker of formula (Va) is provided:
Figure BDA0004113777320000462
or a salt or solvate or stereoisomer thereof; wherein T is a targeting moiety and p is 1 to 20. In some embodiments, p is 1 to 8. In some embodiments, p is 1 to 6. In some embodiments, p is 1 to 4. In some embodiments, p is 2 to 4. In some embodiments, p is 1, 2, 3, or 4.
The present disclosure provides an L1 linker of formula (VI):
Figure BDA0004113777320000463
or a salt or solvate thereof.
The present disclosure provides an L1 linker of formula (VII):
Figure BDA0004113777320000471
or a salt or solvate thereof.
The present disclosure provides an L1 linker of formula (VIII):
Figure BDA0004113777320000472
the present disclosure provides an L1 linker of formula (XII):
Figure BDA0004113777320000473
or a salt or solvate or stereoisomer thereof; wherein R is NO 2 Or NH 2 。
In certain embodiments, L1 linkers can also be generally divided into two classes: cleavable (such as peptide, hydrazone (hydro zone) or disulfide) or non-cleavable (such as thioether). If the linker is a non-cleavable linker, its position on the E3LB moiety should be such that it does not interfere with VHL binding. In particular, the non-cleavable linker is not covalently linked at the hydroxyl position of the proline of the VHL binding domain. Peptide linkers, such as valine-citrulline (Val-Cit), which can be hydrolyzed by lysosomal enzymes, such as cathepsin B, have been used to link drugs to antibodies (US 6,214,345). They are particularly useful, in part, due to their relative stability in the systemic circulation and their ability to release drugs effectively in tumors. However, chemical space represented by natural peptides is limited; it is therefore desirable to have various non-peptide linkers that function like peptides and can be efficiently cleaved by lysosomal proteases. The greater diversity of non-peptide structures can lead to novel beneficial properties not provided by peptide linkers. Provided herein are different types of non-peptide linkers for linker L1 that can be cleaved by lysosomal enzymes.
a. Peptoid linker
Provided herein are different types of non-peptide, peptidomimetic linkers for Ab-CIDE that are cleavable by lysosomal enzymes. For example, an amide bond (e.g., val-Cit) in the middle of a dipeptide is replaced with an amide mimetic; and/or the entire amino acid (e.g., valine amino acid in Val-Cit dipeptide) is replaced with a non-amino acid moiety (e.g., a cycloalkyl dicarbonyl structure (e.g., ring size = 4 or 5)).
When L1 is a peptidomimetic linker, it is represented by the formula
—Str—(PM)—Sp—，
wherein ：
str is a stretcher unit covalently linked to Ab;
sp is a bond or a spacer unit covalently linked to the CIDE moiety; and is also provided with
PM is a non-peptide chemical moiety selected from the group consisting of:
Figure BDA0004113777320000481
w is-NH-heterocycloalkyl-or heterocycloalkyl;
y is heteroaryl, aryl, -C (O) C 1 -C 6 Alkylene group,C 1 -C 6 alkylene-NH 2 、C 1 -C 6 alkylene-NH-CH 3 、C 1 -C 6 alkylene-N- (CH) 3 ) 2 、C 1 -C 6 Alkenyl or C 1 -C 6 An alkylene group;
each R 1 Independently C 1 -C 10 Alkyl, C 1 -C 10 Alkenyl group (C) 1 -C 10 Alkyl) NHC (NH) NH 2 Or (C) 1 -C 10 Alkyl) NHC (O) NH 2 ；
R 3 and R2 Each independently H, C 1 -C 10 Alkyl, C 1 -C 10 Alkenyl, aralkyl or heteroaralkyl, or R 3 and R2 Can together form C 3 -C 7 Cycloalkyl; and is also provided with
R 4 and R5 Each independently is C 1 -C 10 Alkyl, C 1 -C 10 Alkenyl, aralkyl, heteroaralkyl, (C) 1 -C 10 Alkyl) OCH 2 -, or R 4 and R5 Can form C 3 -C 7 Cycloalkyl rings.
Note that L1 may be attached to the CIDE via any of the E3LB, L2 or PB groups.
In embodiments, Y is heteroaryl; r is R 4 and R5 Together forming a cyclobutyl ring.
In an embodiment, Y is a moiety selected from the group consisting of:
Figure BDA0004113777320000491
in embodiments, str is a chemical moiety represented by the formula:
Figure BDA0004113777320000492
wherein R6 Selected from the group consisting of: c (C) 1 -C 10 Alkylene, C 1 -C 10 Alkenyl, C 3 -C 8 Cycloalkyl, (C) 1 -C 8 Alkylene) O-and C 1 -C 10 alkylene-C (O) N (R) a )-C 2 -C 6 An alkylene group, wherein each alkylene group may be substituted with one to five substituents selected from the group consisting of: halo, trifluoromethyl, difluoromethyl, amino, alkylamino, cyano, sulfonyl, sulfonamide, sulfoxide, hydroxy, alkoxy, ester, formic acid, alkylthio, C 3 -C 8 Cycloalkyl, C 4 -C 7 Heterocycloalkyl, aryl, aralkyl, heteroaralkyl, and heteroaryl, each R a Independently H or C 1 -C 6 An alkyl group; sp is-Ar-R b -, wherein Ar is aryl or heteroaryl, R b Is (C) 1 -C 10 Alkylene) O-. Coupling to an antibody can occur when maleimide reacts with an exposed Cys residue on the antibody by michael addition. The exposed Cys residues may be artificially introduced by molecular engineering and/or generated by reduction of interchain disulfide bonds
In embodiments, str has the formula:
Figure BDA0004113777320000493
wherein R7 Selected from C 1 -C 10 Alkylene, C 1 -C 10 Alkenyl group (C) 1 -C 10 Alkylene) O-, N (R) c )-(C 2 -C 6 Alkylene) -N (R) c) and N(Rc )-(C 2 -C 6 An alkylene group); wherein each R is c Independently H or C 1 -C 6 An alkyl group; sp is-Ar-R b -, wherein Ar is aryl or heteroaryl, R b Is (C) 1 -C 10 Alkylene) O-or Sp-C 1 -C 6 alkylene-C (O) NH-.
In an embodiment, L1 is a non-peptide chemical moiety represented by the formula
Figure BDA0004113777320000501
R 1 Is C 1 -C 6 Alkyl, C 1 -C 6 Alkenyl group (C) 1 -C 6 Alkyl) NHC (NH) NH 2 Or (C) 1 -C 6 Alkyl) NHC (O) NH 2 ；
R 3 and R2 Each independently is H or C 1 -C 10 An alkyl group.
In an embodiment, L1 is a non-peptide chemical moiety represented by the formula
Figure BDA0004113777320000502
R 1 Is C 1 -C 6 Alkyl, (C) 1 -C 6 Alkyl) NHC (NH) NH 2 Or (C) 1 -C 6 Alkyl) NHC (O) NH 2 ；
R 4 and R5 Together form C 3 -C 7 Cycloalkyl rings.
In an embodiment, L1 is a non-peptide chemical moiety represented by the formula
Figure BDA0004113777320000503
/>
R 1 Is C 1 -C 6 Alkyl, (C) 1 -C 6 Alkyl) NHC (NH) NH 2 Or (C) 1 -C 6 Alkyl) NHC (O) NH 2 And W is as defined above.
In some embodiments, the linker may be a mimetic peptide linker, the description of which is such as described in WO2015/095227, WO2015/095124, or WO2015/095223, each of which is incorporated herein by reference in its entirety.
In certain embodiments, the linker is selected from the group consisting of:
Figure BDA0004113777320000511
b. non-peptidomimetic linkers
In one aspect, linker L1 may be covalently bound to the antibody and the CIDE as follows:
Figure BDA0004113777320000512
In one aspect, linker L1 forms a disulfide bond with the antibody, and the linker has the structure:
Figure BDA0004113777320000513
wherein R1 、R 2 、R 3 and R4 Independently selected from H, optionally substituted branched or straight chain C 1 -C 5 Alkyl and optionally substituted C 3 -C 6 Cycloalkyl group, or R 1 and R2 Or R is 3 and R4 Together with the carbon atoms to which they are bound form an optionally substituted C 3 -C 6 Cycloalkyl ring or 3-to 6-membered heterocycloalkyl ring.
In one aspect, the carbonyl group of the linker is attached to an amine group in CIDE. It should also be noted that the sulfur atom attached to the Ab is a sulfur group from a cysteine in the antibody. In another aspect, linker L1 has a functional group capable of reacting with free cysteines present on the antibody to form a covalent bond. Non-limiting examples of such reactive functional groups include maleimides, haloacetamides, alpha-haloacetyl, activated esters (e.g., succinimidyl esters), 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. See, e.g., klunssman et al (2004), bioconjugate Chemistry 15 (4): 765-773, page 766, conjugation methods, and examples herein.
In some embodiments, the L1 linker has a functional group capable of reacting with an electrophilic group present on the antibody. Examples of such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some embodiments, a heteroatom of a reactive functional group of a linker may react with an electrophilic group on an antibody and form a covalent bond with an antibody unit. Non-limiting examples of such reactive functional groups include, but are not limited to, hydrazides, oximes, amino groups, hydrazines, thiocarbamides, hydrazinecarboxylic acid esters, and aryl hydrazides.
The L1 linker may comprise one or more linker components. Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl (a "PAB"), N-succinimidyl 4- (2-pyridylthio) pentanoate ("SPP"), and 4- (N-maleimidomethyl) cyclohexane-1 carboxylate ("MCC"). Various linker components are known in the art, some of which are described below.
The L1 linker may be a "cleavable linker" to facilitate release of the CIDE. Non-limiting exemplary cleavable linkers include acid labile linkers (e.g., comprising hydrazone), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, or disulfide-containing linkers (Chari et al, cancer Research 52:127-131 (1992); U.S. Pat. No. 5208020).
In certain embodiments, the linker has the formula:
-A a -W w -Y y -
wherein A is a "drawing unit" and a is an integer from 0 to 1; w is an "amino acid unit" and W is an integer from 0 to 12; y is a "spacer unit" and Y is 0, 1 or 2. An exemplary embodiment of such a joint is described in U.S. patent No. 7,498,298.
In some embodiments, the L1 linker component comprises a "stretch unit" that links the antibody to another linker component or a CIDE moiety. Non-limiting exemplary stretch units are shown below (where wavy lines indicate sites of covalent attachment to antibodies, CIDE, or other linker components):
Figure BDA0004113777320000521
Figure BDA0004113777320000531
/>
in certain embodiments, the linker is:
Figure BDA0004113777320000532
in certain embodiments, the linker has the formula:
—A a —Y y —
wherein A and Y are as defined above. In certain embodiments, the spacer unit Y may be a phosphate, such as a monophosphate or a diphosphate. In certain embodiments, the stretch component a comprises:
Figure BDA0004113777320000533
in certain embodiments, the linker is:
Figure BDA0004113777320000534
3.CIDE(“D”)
useful CIDE has the general formula described above.
Useful Ab-L1-CIDE and unbound degradants exhibit desirable properties such as cell targeting, protein targeting and degradation. In certain embodiments, the Ab-L1-CIDE exhibits a DC50 (μg/mL) of from 0.0001 to less than about 2.0, or less than about 1.0, or less than about 0.8, or less than about 0.7, or less than about 0.6, or less than about 0.5, or less than about 0.4, or less than about 0.3, or less than about 0.2. In certain embodiments, ab-L1-CIDE exhibits a DCmax of at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99.
CIDE includes those having the following composition.
E3 ubiquitin ligase binding group (E3 LB)
E3 ubiquitin ligases (more than 600 are known in humans) confer ubiquitinated substrate specificity. There are known ligands that bind to these ligases. As described herein, an E3 ubiquitin ligase binding group is a peptide or small molecule that can bind E3 ubiquitin ligase as von Hippel-Lindau (VHL).
A specific E3 ubiquitin ligase is von Hippel-Lindau (VHL) tumor suppressor, which is the substrate recognition subunit of the E3 ligase complex VCB, which also consists of elongation proteins B and C, cul and Rbxl. The primary substrate for VHL is hypoxia inducible factor lα (HIF-lα), a transcription factor that can up-regulate genes in response to hypoxia levels, such as the angiogenic growth factor VEGF and the erythrocyte-induced cytokine erythropoietin.
In one aspect, the subject matter herein relates to the E3LB part of CIDE, which has the following chemical structure:
Figure BDA0004113777320000541
wherein ,R1A 、R 1B and R1C Each independently is hydrogen or C 1-5 An alkyl group; or R is 1A 、R 1B and R1C Two of which together with the carbon to which each is attached form C 1-5 Cycloalkyl;
R 2 is C 1-5 An alkyl group;
R 3 selected from the group consisting of cyano groups,
Figure BDA0004113777320000551
A group of>
Figure BDA0004113777320000554
Is a single bond or a double bond; and q is 1 or 0;
Y 1 and Y2 One of them is-CH, Y 1 and Y2 The other of them is-CH or N;
wherein L1-T, L1-U, L1-V and L1-Y are each independently as described elsewhere herein; and L2 is as described elsewhere herein.
In certain embodiments, E3LB has wherein R 3 Is cyano structure.
In certain embodiments, E3LB has wherein R 3 Is that
Figure BDA0004113777320000552
Is a structure of (a).
In certain embodiments, E3LB has wherein R 3 Is that
Figure BDA0004113777320000553
Is a structure of (a).
In certain embodiments, E3LB has wherein R 1A 、R 1B and R1C Each independently is hydrogen or methyl.
In certain embodiments, E3LB has wherein R 1A and R1B Each of which is methyl.
In certain embodiments, E3LB has one of the following formulas:
Figure BDA0004113777320000561
Figure BDA0004113777320000571
in certain embodiments, E3LB has wherein R 2 Is hydrogen, methyl, ethyl or propyl.
In certain embodiments, E3LB has wherein R 2 Is a methyl group.
In certain embodiments, E3LB has wherein R 2 Is that
Figure BDA0004113777320000574
Is a structure of (a).
In certain embodiments, E3LB has wherein Y 1 and Y2 Each of them has a structure of-CH.
In certain embodiments, E3LB has wherein Y 1 Is N and Y 2 Is the structure of-CH.
In certain embodiments, E3LB has wherein Y 1 is-CH and Y 2 Is a structure of N.
In certain embodiments, the proline portion of E3LB has the following structure:
Figure BDA0004113777320000573
The E3LB moiety has at least one terminus with a moiety covalently linked or covalently connectable to the L2 moiety and at least one terminus with a moiety covalently linked or covalently connectable to the L1 moiety. For example, the E3LB moiety terminates in a-NHCOOH moiety, which may be covalently linked to the L2 moiety through an amide bond.
In any aspect or embodiment described herein, E3LB described herein may be a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, or polymorph thereof. In addition, in any aspect or embodiment described herein, E3LB described herein may be coupled to PB directly via a bond or via a chemical linker.
BRM protein binding group (PB)
The PB portion of CIDE is the portion of the small molecule that binds to BRM, including all variants, mutations, splice variants, insertions/deletions and fusions of BRM. BRMs are also known as subfamily a, member 2, SMARCA2 and BRAHMA. Such small molecule target protein binding moieties also include pharmaceutically acceptable salts, enantiomers, solvates, and polymorphs of these compositions, as well as other small molecules that can target the target protein.
The CIDE or DAC described herein may comprise any residue of known BRM binding compounds, including those disclosed in WO2019/195201, which is incorporated herein by reference in its entirety.
In certain embodiments, the BRM-binding compound is a compound of formula I:
Figure BDA0004113777320000581
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any of the foregoing, wherein:
wherein X is hydrogen or halogen;
Figure BDA0004113777320000582
selected from the group consisting of: />
Figure BDA0004113777320000591
Wherein for (a) to (e), represents the point of attachment to [ X ] or, if [ X ] is absent, the point of attachment to [ Y ] and the point of attachment to the phenyl ring; and wherein:
(i) [ X ] is a 3-to 15-membered heterocyclic group or a 5-to 20-membered heteroaryl group,
provided that when
Figure BDA0004113777320000601
In the case of (a), then [ X ]]Not->
Figure BDA0004113777320000602
Figure BDA0004113777320000603
Wherein # represents->
Figure BDA0004113777320000604
And # denotes the connection point with L2,
[ Y ] is absent, and
[ Z ] is absent; or alternatively
(ii)[X]Is a 3-to 15-membered heterocyclyl or a 5-to 20-membered heteroaryl, wherein [ X ]]Optionally substituted 3-to 15-membered heterocyclyl with one or more-OH or C 1-6 An alkyl group is substituted and a substituent is substituted,
[ Y ] is absent, and
[ Z ] is a 3-to 15-membered heterocyclic group or a 5-to 20-membered heteroaryl group,
provided that when
Figure BDA0004113777320000605
Is (a) and [ X ]]Is->
Figure BDA0004113777320000606
In which&Representation and->
Figure BDA0004113777320000607
And (2) connection point of&&Representation and [ Z ]]And [ Z ]]Not->
Figure BDA0004113777320000608
Wherein # denotes an integer of and [ X ]]And # denotes the connection point to L2; or alternatively
(iii) [ X ] is a 3-to 15-membered heterocyclic group or a 5-to 20-membered heteroaryl group,
[ Y ] is a methylene group, wherein the methylene group of [ Y ] is optionally substituted with one or more methyl groups, and
[ Z ] is a 3-to 15-membered heterocyclic group; or alternatively
(iv) [ X ] is not present and,
[ Y ] is a vinylidene group, wherein the vinylidene group of [ Y ] is optionally substituted with one or more halo groups, and
[ Z ] is a 5-to 20-membered heteroaryl,
provided that the conditions are that,
Figure BDA0004113777320000609
is (a), (b), (d) or (e); or alternatively
(v) [ X ] is not present and,
[ Y ] is ethynylene, and
[ Z ] is a 5-to 20-membered heteroaryl,
provided that the conditions are that,
Figure BDA0004113777320000611
is (a), (b), (d) or (e); or alternatively
(vi) [ X ] is not present and,
[ Y ] is cyclopropyl or cyclobutyl, and
[ Z ] is a 5-to 20-membered heteroaryl,
provided that the conditions are that,
Figure BDA0004113777320000612
is (a), (b), (d) or (e).
In certain embodiments, the BRM-binding compound is a compound of formula (I-a):
Figure BDA0004113777320000613
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any of the foregoing, wherein X is hydrogen or halo, and wherein [ X ], [ Y ] and [ Z ] are as defined above for the compound of formula (I).
In certain embodiments, the BRM-binding compound is a compound of formula (I-B):
Figure BDA0004113777320000614
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any of the foregoing, wherein X is hydrogen or halo, and wherein [ X ], [ Y ] and [ Z ] are as defined above for the compound of formula (I).
In certain embodiments, the BRM-binding compound is a compound of formula (I-C):
Figure BDA0004113777320000621
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any of the foregoing, wherein X is hydrogen or halo, and wherein [ X ], [ Y ] and [ Z ] are as defined above for the compound of formula (I).
In certain embodiments, the BRM-binding compound is a compound of formula (I-D):
Figure BDA0004113777320000622
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any of the foregoing, wherein X is hydrogen or halo, and wherein [ X ], [ Y ] and [ Z ] are as defined above for the compound of formula (I).
In certain embodiments, the BRM-binding compound is a compound of formula (I-E):
Figure BDA0004113777320000623
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any of the foregoing, wherein X is hydrogen or halo, and wherein [ X ], [ Y ] and [ Z ] are as defined above for the compound of formula (I).
In certain embodiments, the PB (BRM) portion of CIDE has the following structure:
Figure BDA0004113777320000631
wherein ,
Figure BDA0004113777320000632
is a covalent attachment point to L2.
c. Joint L2
The E3LB and PB portions of CIDE as described herein may be linked to linkers (L2, linker-2). In certain embodiments, linker L2 is covalently bound to the E3LB moiety and to the PB moiety, thereby constituting the cime.
In certain embodiments, the L2 moiety may be selected from the linkers disclosed in WO2019/195201, which is incorporated herein by reference in its entirety.
Although the E3LB and PB groups may be covalently linked to the linker through any group suitable and stable to the chemical nature of the linker, in certain aspects L2 is covalently bound independently to the E3LB and PB groups through an amide, ester, thioester, keto, urethane (urethane) or ether, each group may be inserted anywhere on the E3LB and PB groups to allow binding of the E3LB group to ubiquitin ligase and the PB group to the BRM target protein to be degraded. In other words, as shown herein, linkers may be designed and linked to E3LB and PB to regulate binding of E3LB and PB to their respective binding partners.
In certain embodiments, L2 is a linker covalently bound to E3LB and PB, the L2 having the formula:
Figure BDA0004113777320000641
wherein ,
R 4 is hydrogen or methyl, and is preferably hydrogen or methyl,
Figure BDA0004113777320000642
wherein ,
z is one or zero and is zero,
g is
Figure BDA0004113777320000643
or-C (O) NH-; and is also provided with
Figure BDA0004113777320000651
Is the point of attachment to PB.
In certain embodiments of L2a, R 4 Is hydrogen.
In certain embodiments of L2a, R 4 Is methyl.
In certain embodiments of L2a, R 4 Is methyl, such that the methyl group is positioned relative to the piperazine to which it is attached as follows:
Figure BDA0004113777320000652
In certain embodiments of L2c, z is zero.
In certain embodiments of L2c, z is one.
Referring now to Ab-CIDE, ab-CIDE can be packagedComprises a single antibody, wherein the single antibody may have more than one CIDE, each CIDE being covalently linked to the antibody through a linker L1. "CIDE load" is the average number of CIDE moieties per antibody. The CIDE loading per antibody (Ab) may range from 1 to 20 CIDE (D). Namely, in the Ab-CIDE formula, ab-is (L1-D) p In which p has a value of from about 1 to about 20, from about 1 to about 8, from about 1 to about 5, from about 1 to about 4, or from about 1 to about 3. Each of the CIDEs covalently linked to the antibody through linker L1 may be the same or different CIDEs, and may have the same or different types of linkers as any other L1 covalently linked to the antibody. In certain embodiments, ab is a cysteine engineered antibody and p is about 2.
The average amount of CIDE per antibody in the Ab-CIDE formulation obtained by conjugation reaction can be characterized by conventional methods such as mass spectrometry, ELISA assay, electrophoresis and HPLC. Quantitative distribution of Ab-CIDE expressed as p can also be determined. The average value of p in a particular Ab-CIDE formulation can be determined by ELISA (Hamblett et al (2004) Clin. Cancer Res.10:7063-7070; sanderson et al (2005) Clin. Cancer Res. 11:843-852). However, the distribution of p-values cannot be discerned by the limitations of antibody-antigen binding and detection by ELISA. Also, ELISA assays for detecting Ab-CIDE cannot determine where the CIDE moiety is attached to an antibody, such as a heavy or light chain fragment or a specific amino acid residue. In some cases, isolation, purification and characterization of homogeneous Ab-CIDE where p is a particular value obtained from Ab-CIDE with other CIDE loadings can be achieved by reverse phase HPLC or electrophoresis, among other methods.
For some Ab-CIDE, p may be limited by the number of binding sites on the antibody. For example, an antibody may have only one or a few cysteine thiol groups, or may have only one or a few thiol groups with sufficiently high reactivity through which a linker may be attached. Another reactive site on Ab for attachment of L1-D is the amine functionality of a lysine residue. p values include values from about 1 to about 20, from about 1 to about 8, from about 1 to about 5, from about 1 to about 4, from about 1 to about 3, where p is equal to 2. In some embodiments, the subject matter described herein is directed to any Ab-CIDE, wherein p is about 1, 2, 3, 4, 5, 6, 7, or 8.
Typically, less than the theoretical maximum of CIDE moieties are conjugated to the antibody during the conjugation reaction. Antibodies may comprise, for example, a number of lysine residues that do not react with the linker L1-CIDE group (L1-D) or the linker reagent. Only the most reactive lysine groups may react with the amine reactive linker. Furthermore, only the most reactive cysteine thiol groups can react with thiol-reactive linkers or linker L1-CIDE groups. Typically, antibodies do not contain many, if any, free and reactive cysteine thiol groups that can be attached to the CIDE moiety. Most of the cysteine thiol residues in compound antibodies exist as disulfide bonds and must be reduced with a reducing agent such as Dithiothreitol (DTT) or TCEP under partial or complete reducing conditions. However, the CIDE loading of the CAR (CIDE/antibody ratio, "CAR") can be controlled in several different ways, including: (i) limit the molar excess of linker L1-CIDE group or linker agent relative to the antibody, (ii) limit the conjugation reaction time or temperature, and (iii) reduce conditions that partially or limit cysteine thiol modification.
L1-CIDE Compounds
The CIDE described herein may be covalently linked to linker L1 to produce an L1-CIDE group. These compounds have the general formula:
(L1―D)，
wherein D is CIDE having the structure E3 LB-L2-PB; wherein E3LB is an E3 ligase binding group covalently bound to L2. L2 is a linker covalently bound to E3LB and PB; PB is a BRM protein binding group that is covalently bound to L2. And L1 is a linker covalently bound to D. Useful groups for each of these components are as described above.
In certain embodiments, L1 is as described elsewhere herein, including a peptidomimetic linker. In these embodiments, L1-CIDE has the formula:
Figure BDA0004113777320000661
wherein
Str is a stretch unit;
sp is a bond or a spacer unit covalently linked to the D, CIDE moiety;
R 1 is C 1 -C 10 Alkyl, (C) 1 -C 10 Alkyl) NHC (NH) NH 2 Or (C) 1 -C 10 Alkyl) NHC (O) NH 2 ；
R 4 and R5 Each independently is C 1 -C 10 Alkyl, aralkyl, heteroaralkyl, (C) 1 -C 10 Alkyl) OCH 2-, or R4 and R5 Can form C 3 -C 7 A cycloalkyl ring;
d is the CIDE part.
The L1-CIDE compound may be represented by the formula:
Figure BDA0004113777320000671
wherein R6 Is C 1 -C 10 An alkylene group; r is R 4 and R5 Together form C 3 -C 7 Cycloalkyl ring, and D is the CIDE moiety.
The L1-CIDE compound may be represented by the formula:
Figure BDA0004113777320000672
wherein R1 、R 4 and R5 As described elsewhere herein, and D is the ciae moiety.
The L1-CIDE compound may be represented by the formula:
Figure BDA0004113777320000673
wherein
Str is a stretch unit;
sp is an optional spacer unit covalently linked to the D, CIDE moiety;
y is heteroaryl, aryl, -C (O) C 1 -C 6 Alkylene group,C 1 -C 6 alkylene-NH 2 、C 1 -C 6 alkylene-NH-CH 3 、C 1 -C 6 alkylene-N- (CH) 3 ) 2 、C 1 -C 6 Alkenyl or C 1 -C 6 An alkylene group;
R 1 is C 1 -C 10 Alkyl, (C) 1 -C 10 Alkyl) NHC (NH) NH 2 Or (C) 1 -C 10 Alkyl) NHC (O) NH 2 ；
R 3 and R2 Each independently is H, C 1 -C 10 Alkyl, aralkyl or heteroaralkyl, or R 3 and R2 Can together form C 3 -C 7 Cycloalkyl; and is also provided with
D is the CIDE part.
The L1-CIDE compound may be represented by the formula:
Figure BDA0004113777320000681
wherein R6 Is C 1 -C 10 Alkylene group, and R 1 、R 2 and R3 As described elsewhere herein, and D is part CIDE
The L1-CIDE compound may be represented by the formula:
Figure BDA0004113777320000682
wherein R1 、R 2 and R3 As described elsewhere herein, and D is the ciae moiety.
In any of the above L1-CIDE compounds, str may have the formula:
Figure BDA0004113777320000683
wherein R6 Selected from C 1 -C 10 Alkylene, C 3 -C 8 Cycloalkyl, O- (C) 1 -C 8 Alkylene) and C 1 -C 10 alkylene-C (O) N (R) a )-C 2 -C 6 An alkylene group, wherein each alkylene group may be substituted with one to five substituents selected from the group consisting of: halo, trifluoromethyl, difluoromethyl, amino, alkylamino, cyano, sulfonyl, sulfonamide, sulfoxide, hydroxy, alkoxy, ester, formic acid, alkylthio, C 3 -C 8 Cycloalkyl, C 4 -C 7 Heterocycloalkylaryl, aralkyl, heteroaralkyl, and heteroaryl; each R a Independently H or C 1 -C 6 An alkyl group; sp is-Ar-R b -, wherein Ar is aryl or heteroaryl, R b Is (C) 1 -C 10 Alkylene) O-.
In certain L1-CIDE compounds, R 6 Is C 1 -C 10 Alkylene, sp is-Ar-R b -, wherein Ar is aryl, R b Is (C) 1 -C 6 Alkylene) O-; or R is 6 Is- (CH) 2 ) q 1-10;
in any of the above L1-CIDE compounds, str may have the formula:
Figure BDA0004113777320000691
wherein ,
Figure BDA0004113777320000692
represents a moiety capable of conjugation to an antibody, R 7 Selected from C 1 -C 10 Alkylene, C 1 -C 10 alkylene-O, N (R) c )-(C 2 -C 6 Alkylene) -N (R) c) and N(Rc )-(C 2 -C 6 Alkylene) groups; wherein each R is c Independently H or C 1 -C 6 An alkyl group; />
Sp is-Ar-R b -, wherein Ar is aryl or heteroaryl, R b Is (C) 1 -C 10 Alkylene) O-; or wherein R is 6 Is C 1 -C 10 Alkylene group, sp is-Ar-R b Wherein Ar is aryl, R b Is (C) 1 -C 6 Alkylene) O-.
L1-CIDE may have the formula, wherein in each case D is the CIDE part:
Figure BDA0004113777320000693
and
Figure BDA0004113777320000701
Referring now to the PB radical of CIDE, in particular embodiments PB is as described elsewhere herein. Referring now to the E3LB group of CIDE, E3LB is as described elsewhere herein. Ab-CIDE can include any combination of PB, E3LB, ab, L1, and L2.
Those skilled in the art will appreciate in view of the subject matter disclosed herein that the point of attachment of L1 and L2 may vary. In addition, partial linkers, such as-Str- (PM) -Sp-may be interchanged. In addition, the partial joints L1 may be interchanged. Non-limiting examples of L1 linkers attached to CIDE, antibodies, and other interchangeable linkers include, but are not limited to, those shown in tables 1-L1.
Figure BDA0004113777320000702
/>
Figure BDA0004113777320000711
/>
Figure BDA0004113777320000721
/>
Figure BDA0004113777320000731
/>
Figure BDA0004113777320000741
In certain embodiments, the linker L1 may be at different positions L1-T, L1-U, L1-V and L1-Y (from R 3 Group) to an E3LB residue:
Figure BDA0004113777320000751
/>
R 3 selected from the group consisting of cyano groups,
Figure BDA0004113777320000752
A group of which, wherein,
Figure BDA0004113777320000753
is a single or double bond;
ab is an antibody covalently bound to at least one L1, L1 being a linker;
L1-T, L1-U and L1-V are each independently hydrogen or an L1 linker covalently bound to Ab and D;
L1-Y is hydrogen or an L1 linker covalently bound to Ab and D; and is also provided with
q is 1 or 0.
The linker L1 may be attached to any position of the antibody as long as the covalent bond between the linker L1 and the antibody is a disulfide bond.
In embodiments, the antibodies Ab are each conjugated to one to eight chemical degradation inducers (CIDE) D via a linker L1.
Ab―(L1―D) p Wherein p is 1 to 8
D comprises an E3 ligase binding (E3 LB) ligand linked to a target Protein Binding (PB) ligand by a linker L2 as follows:
E3LB—L2—PB
in an embodiment, L1 forms a disulfide bond with the sulfur of the engineered Cys residue of the antibody to link the CIDE to the Ab.
In an embodiment, the antibody is linked to the E3LB ligand of CIDE via L1.
In an embodiment, L1 is linked to an E3LB ligand residue of an E3LB ligand of CIDE.
For example, in an embodiment, L1 is covalently bound to a portion of the BRM at the point of attachment (L1-Q), as follows:
Figure BDA0004113777320000761
Wherein X is hydrogen or halo and L1 is selected from L1b and L1c.
In an embodiment, L1 is covalently bound to a portion of the BRM at the point of attachment (L1-Q'), as follows:
Figure BDA0004113777320000762
wherein X is hydrogen or halo and L1 is selected from L1b and L1c.
In an embodiment, L1 is covalently bound to a portion of E3LB at the point of attachment (L1-Q'), as follows:
Figure BDA0004113777320000763
wherein L1 is selected from L1a, L1b and L1c.
Reference is now made to Ab-CIDE and L1-CIDE compounds described herein, which may be present in solid or liquid form. In the solid state, it may exist in crystalline or non-crystalline form or as a mixture thereof. The skilled artisan will appreciate that pharmaceutically acceptable solvates for crystalline or non-crystalline compounds may be formed. In crystalline solvates, solvent molecules are incorporated into the crystal lattice during crystallization. Solvates may involve non-aqueous solvents such as, but not limited to, ethanol, isopropanol, DMSO, acetic acid, ethanolamine, or ethyl acetate, or they may involve water as a solvent for incorporation into the crystal lattice. Solvates in which water is the solvent incorporated into the crystal lattice are commonly referred to as "hydrates". Hydrates include stoichiometric hydrates and compositions containing variable amounts of water. The subject matter described herein includes all such solvates.
Those of skill in the art will further appreciate that certain of the compounds described herein in crystalline form and Ab-CIDE, including various solvates thereof, may exhibit polymorphism (i.e., the ability to occur in different crystal structures). These different crystal forms are commonly referred to as "polymorphs". The subject matter disclosed herein includes all such polymorphs. Polymorphs have the same chemical composition but differ in packing, geometric arrangement and other descriptive properties of the crystalline solid state. Thus, polymorphs may have different physical properties such as shape, density, hardness, deformability, stability and dissolution properties. Polymorphs typically exhibit different melting points, IR spectra and X-ray powder diffraction patterns, which can be used for identification. Those skilled in the art will appreciate that different polymorphs may be produced, for example, by varying or adjusting the reaction conditions or reagents used to prepare the compounds. For example, changes in temperature, pressure, or solvent may result in polymorphs. In addition, under certain conditions, one polymorph may spontaneously convert to another polymorph.
The compounds described herein and Ab-CIDE or salts thereof may exist in stereoisomeric forms (e.g., which contain one or more asymmetric carbon atoms). Various stereoisomers (enantiomers and diastereomers) and mixtures thereof are included within the scope of the subject matter disclosed herein. Also, it is to be understood that the compounds or salts of formula (I) may exist in tautomeric forms other than the structures shown in the formulae, and are also included within the scope of the subject matter disclosed herein. It should be understood that the subject matter disclosed herein includes all combinations and subsets of the specific set forth herein. The subject matter disclosed herein includes mixtures of stereoisomers and purified enantiomers or enantiomerically/diastereomerically enriched mixtures. It is to be understood that the subject matter disclosed herein includes all combinations and subsets of the specific set forth herein above.
The subject matter disclosed herein also includes isotopically-labeled forms of the compounds described herein, but in fact, one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds described herein and pharmaceutically acceptable salts thereofIsotopes including hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H、 3 H、 11 C、 13 C、 14 C、 15 N、 17 O、 18 O、 31 P、 32 P、 35 S、 18 F、 36 Cl、 123I and 125 I。
compounds disclosed herein and Ab-CIDE and pharmaceutically acceptable salts thereof comprising the foregoing isotopes and/or other isotopes of other atoms are within the scope of the subject matter disclosed herein. Isotopically-labeled compounds are disclosed herein, e.g., having incorporated therein a radioisotope (e.g 3 H、 14 C) Can be used in drug and/or substrate tissue distribution assays. Tritiation in general (i.e 3 H) And carbon-14 (i.e 14 C) Isotopes because of their ease of preparation and detection. 11C and 18 The F isotope can be used for PET (positron emission tomography), while 125 The I isotope can be used for SPECT (single photon emission computed tomography), all of which can be used for brain imaging. Further, the use of heavier isotopes such as deuterium (i.e., 2 h) Substitution may provide certain therapeutic advantages due to higher metabolic stability (e.g., increased in vivo half-life or dose reduction requirements) and thus may be preferred in certain circumstances. Isotopically-labeled compounds can generally be prepared by performing the procedures disclosed in the schemes and/or examples below (by substituting a non-isotopically-labeled reagent for a readily available isotopically-labeled reagent).
In some embodiments, D is
Figure BDA0004113777320000781
Wherein L1 is covalently linked to D at one point of attachment selected from the group consisting of: L1-Q, L1-Q', L1-S, L1-T, L1-U, L1-V and L1-Y. It should be appreciated that each of the attachment points other than L1 in L1-Q, L1-Q', L1-S, L1-T, L1-U, L1-V and L1-Y retain their original valence state. For example, if L1 is attached at L1-Q', it is not attached at L1-Q, L1-S, L1-T, L1-U, L1-V or L1-Y, and D has the structure:
Figure BDA0004113777320000782
in certain embodiments, L1 is L1a, which has the structure:
Figure BDA0004113777320000791
R a 、R b 、R c and Rd Each independently selected from the group consisting of H, optionally substituted branched or straight chain C 1 -C 5 Alkyl and optionally substituted C 3 -C 6 Cycloalkyl group, or R a and Rb Or R is c and Rd Together with the carbon atoms to which they are bound form an optionally substituted C 3 -C 6 Cycloalkyl ring or 3-to 6-membered heterocycloalkyl ring, and wherein
Figure BDA0004113777320000792
Is the point of attachment to Ab.
In embodiments, L1a is attached at L1-T, and R a 、R b 、R c and Rd At least one of which is methyl.
In embodiments, L1a is attached at L1-T, and R a 、R b 、R c and Rd At least two of which are methyl groups.
In embodiments, L1a is attached at L1-T, and R a and Rc Each is methyl, and R b and Rd Each hydrogen.
In embodiments, L1a is attached at L1-T, and R a 、R c and Rd Each is methyl, and R b Is hydrogen.
In embodiments, L1a is attached at L1-T, and R a and Rb Each is hydrogen, and R c and Rd Combined with themIs bonded to form an optionally substituted 3-to 6-membered heterocycloalkyl ring. In embodiments, the 3-to 6-membered heterocycloalkyl ring is an optionally substituted piperidine ring. In an embodiment, the piperidine ring is substituted with methyl.
In an embodiment, L1a is attached at L1-T, wherein R a 、R b 、R c and Rd At least two of which are methyl groups; and a phosphate moiety is attached at L1-Q, wherein the phosphate moiety has the structure:
Figure BDA0004113777320000793
wherein e is 1.
In certain embodiments, L1 is L1b, which has the structure:
Figure BDA0004113777320000801
wherein Z and Z 1 Each independently is C 1-12 Alkylene or- - [ CH ] 2 ] g -[-O-CH 2 ] h -, wherein g is 0, 1 or 2, and h is 1 to 5; r is R z Is H or C 1-3 An alkyl group; d is 0, 1 or 2; and wherein->
Figure BDA0004113777320000802
Is the point of attachment to Ab.
In embodiments, Z and Z 1 Each independently is C 1-12 Alkylene group, R z Is hydrogen and d is 0 or 1.
In embodiments, Z is C 2 Alkylene group, and Z 1 Is C 5 Alkylene group, R z Is hydrogen and d is 0 or 1.
In an embodiment, L1b is attached at L1-Q, and d is 1.
In an embodiment, L1b is attached at L1-T, and d is 0.
In certain embodiments, L1 is L1c, which has the structure:
Figure BDA0004113777320000803
wherein
Z 2 Is C 1-12 Alkylene or- [ CH ] 2 ] g -[-O-CH 2 ] h -, wherein g is 0, 1 or 2, and h is 1 to 5;
w is 0, 1, 2, 3, 4 or 5, and wherein
Figure BDA0004113777320000804
Is the point of attachment to Ab;
j is hydrogen, -N (R) x )(R y )、–C(O)NH 2 、–NH-C(O)-NH 2 、–NH-C(＝NH)-NH 2, wherein Rx and Ry Each independently selected from hydrogen and C 1-3 Alkyl, wherein R is x and Ry Each independently selected from hydrogen and C 1-3 An alkyl group;
k is selected from-CH 2 –、–CH(R)–、–CH(R)-O–^、–C(O)–、^–C(O)-O-CH(R)–、–CH 2 -O-C(O)–^、–CH 2 -O-C(O)-NH-^、^-O-C(L1c)-C(O)-NR x R y -、^-C(L1c)-C(O)-NR x R y -、-CH 2 -O-C(O)-NH-CH 2 –、–CH 2 -O-C(O)-R-[CH 2 ] q -O–^、–CH 2 -O-C(O)-R-[CH 2 ] q -the process comprises, where a represents the connection to CIDE, wherein R is hydrogen, C 1-3 Alkyl, N (R) x )(R y )、–O-N(R x )(R y ) Or C (O) -N (R) x )(R y ) Wherein q is 0, 1, 2 or 3, and R x and Ry Each independently selected from hydrogen and C 1-3 Alkyl, or R x and Ry Together with the nitrogen to which each is attached, form an optionally substituted 5-to 7-membered heterocyclyl;
ra and Rb are each independently selected from hydrogen and C 1-3 Alkyl, or Ra and Rb, together with the carbon to which each is attached, form an optionally substituted C 3-6 Cycloalkyl; and
R 7 and R8 Each independently is hydrogen, halo, C 1-5 Alkyl, C 1-5 Alkoxy or hydroxy.
In an embodiment, Z 2 Is C 1-12 Alkylene, w is 2, J is-NH-C (O) -NH 2 Ra and Rb, together with the carbon to which each is attached, form an optionally substituted C 3-6 Cycloalkyl, and R 7 and R8 Each independently is hydrogen.
In an embodiment, Z 2 Is C 5 Alkylene, w is 2, J is-NH-C (O) -NH 2 Ra and Rb, together with the carbon to which each is attached, form an optionally substituted C 4 Cycloalkyl, and R 7 and R8 Each independently is hydrogen.
In an embodiment, Z 2 Is C 1-12 Alkylene, w is 2, J is-NH-C (O) -NH 2 K is-CH 2 -O-C (O) -, ra and Rb together with the carbon to which each is attached form an optionally substituted C 3-6 Cycloalkyl, and R 7 and R8 Each independently is hydrogen.
In an embodiment, Z 2 Is C 5 Alkylene, w is 2, J is-NH-C (O) -NH 2 K is-CH 2 -O-C (O) -, ra and Rb together with the carbon to which each is attached form an optionally substituted C 4 Cycloalkyl, and R 7 and R8 Each independently is hydrogen.
In an embodiment, Z 2 Is C 1-12 Alkylene, w is 3, J is-N (R x )(R y), wherein Rx and Ry Each independently selected from hydrogen and C 1-3 Alkyl, ra and Rb together with the carbon to which each is attached form an optionally substituted C 3-6 Cycloalkyl, and R 7 and R8 Each independently is hydrogen.
In an embodiment, Z 2 Is C 5 Alkylene, w is 3, J is-N (R x )(R y), wherein Rx and Ry Each methyl, ra and Rb, together with the carbon to which each is attached, form C 4 Cycloalkyl, and R 7 and R8 Each independently is hydrogen.
In an embodiment, Z 2 Is C 1-12 Alkylene, w is 3, J is-N (R x )(R y), wherein Rx and Ry Each independently selected from hydrogen and C 1-3 Alkyl, K is-CH 2 -, ra and Rb together with the carbon to which each is attached form an optionally substituted C 3-6 Cycloalkyl, and R 7 and R8 Each independently is hydrogen.
In an embodiment, Z 2 Is C 5 Alkylene, w is 3, J is-N (R x )(R y), wherein Rx and Ry Each is methyl, K is-CH 2 -, ra and Rb together with the carbon to which each is attached form C 4 Cycloalkyl, and R 7 and R8 Each independently is hydrogen.
In an embodiment, Z 2 Is C 1-12 Alkylene, w is 0, J is hydrogen, ra and Rb, together with the carbon to which each is attached, form an optionally substituted C 3-6 Cycloalkyl, and R 7 and R8 Each independently is hydrogen.
In an embodiment, Z 2 Is C 5 Alkylene, w is 0, J is hydrogen, ra and Rb together with the carbon to which each is attached form C 4 Cycloalkyl, and R 7 and R8 Each independently is hydrogen.
In an embodiment, Z 2 Is C 1-12 Alkylene, w is 0, J is hydrogen, K is-CH 2 -, ra and Rb together with the carbon to which each is attached form an optionally substituted C 3-6 Cycloalkyl, and R 7 and R8 Each independently is hydrogen.
In an embodiment, Z 2 Is C 5 Alkylene, w is 0, J is hydrogen, K is-CH 2 -, ra and Rb together with the carbon to which each is attached form C 4 Cycloalkyl, and R 7 and R8 Each independently is hydrogen.
In an embodiment, Z 2 Is C 1-12 Alkylene, w is 2, J is-NH-C (O) -NH 2 Ra and Rb, together with the carbon to which each is attached, form an optionally substituted C 3-6 Cycloalkyl, and R 7 and R8 Each independently is hydrogen.
In an embodiment, Z 2 Is C 5 Alkylene, w is 2, J is-NH-CO)-NH 2 Ra and Rb, together with the carbon to which each is attached, form an optionally substituted C 4 Cycloalkyl, and R 7 and R8 Each independently is hydrogen.
In an embodiment, Z 2 Is C 1-12 Alkylene, w is 2, J is-NH-C (O) -NH 2 K is-CH (R) -O-C (O) -, wherein R is C (O) -N (R) x )(R y), wherein Rx and Ry Together with the nitrogen to which each is attached form an optionally substituted 5-to 7-membered heterocyclyl, ra and Rb together with the carbon to which each is attached form an optionally substituted C 3-6 Cycloalkyl, and R 7 and R8 Each independently is hydrogen.
In an embodiment, Z 2 Is C 5 Alkylene, w is 2, J is-NH-C (O) -NH 2 K is-CH (R) -O-C (O) -, wherein R is C (O) -N (R) x )(R y), wherein Rx and Ry Together with the nitrogen to which each is attached, form an optionally substituted piperazine, ra and Rb together with the carbon to which each is attached form an optionally substituted C 4 Cycloalkyl, and R 7 and R8 Each independently is hydrogen.
In an embodiment, Z 2 Is C 1-12 Alkylene, w is 3, J is-N (R x )(R y), wherein Rx and Ry Each independently selected from hydrogen and C 1-3 Alkyl, K is-CH 2 -O-C (O) -, ra and Rb together with the carbon to which each is attached form an optionally substituted C 3-6 Cycloalkyl, and R 7 and R8 Each independently is hydrogen.
In an embodiment, Z 2 Is C 5 Alkylene, w is 3, J is-N (R x )(R y), wherein Rx and Ry Each is methyl, K is-CH 2 -O-C (O) -, ra and Rb, together with the carbon to which each is attached, form C 4 Cycloalkyl, and R 7 and R8 Each independently is hydrogen.
In an embodiment, L1c is attached at L1-Q and K is-CH 2 –。
In an embodiment, L1c is attached at L1-Q',and K is-CH 2 -O-C(O)–。
In an embodiment, L1c is attached at L1-S and K is-CH 2 –。
In an embodiment, L1C is attached at L1-T, and K is-CH (R) -O-C (O) -, where R is C (O) -N (R) x )(R y), wherein Rx and Ry Together with the nitrogen to which each is attached, form an optionally substituted 5-to 7-membered heterocyclyl.
In an embodiment, L1c is attached at L1-U.
In an embodiment, L1c is attached at L1-V.
In an embodiment, L1c is attached at L1-Y and K is-CH 2 –。
In an embodiment, L1c is attached at L1-Q, where K is-CH 2 -; and a phosphate moiety is attached at L1-T, wherein the phosphate moiety has the structure
Figure BDA0004113777320000831
Wherein e is 0.
In certain embodiments, the subject matter described herein includes the following L1-CIDE.
Figure BDA0004113777320000832
/>
Figure BDA0004113777320000841
/>
Figure BDA0004113777320000851
/>
Figure BDA0004113777320000861
The subject matter disclosed herein includes the following non-limiting examples:
1. conjugate with the following chemical structure
Ab―(L1―D) p ，
wherein ,
d is CIDE with E3 LB-L2-PB structure;
e3LB is covalently bound to L2, said E3LB having the formula:
Figure BDA0004113777320000871
wherein ,
R 1A 、R 1B and R1C Each independently is hydrogen or C 1-5 An alkyl group; or R is 1A 、R 1B And
R 1C two of which together with the carbon to which each is attached form C 1-5 Cycloalkyl;
R 2 is C 1-5 An alkyl group;
R 3 selected from the group consisting of cyano groups,
Figure BDA0004113777320000872
A group of which, wherein,----is a single bond or a double bond;
Y 1 and Y2 One of them is-CH, Y 1 and Y2 The other of them is-CH or N;
l2 is a linker covalently bound to E3LB and PB, said L2 having the formula:
Figure BDA0004113777320000873
wherein ,
R 4 is hydrogen or methyl, and is preferably hydrogen or methyl,
Figure BDA0004113777320000881
/>
wherein ,
z is one or zero and is zero,
g is
Figure BDA0004113777320000882
or-C (O) NH-; and is also provided with
Figure BDA0004113777320000883
Is a connection point with PB
PB is a protein binding group covalently bound to L2, which has the following structure:
Figure BDA0004113777320000884
Figure BDA0004113777320000891
ab is an antibody covalently bound to at least one L1, L1 being a linker;
L1-T, L1-U and L1-V are each independently hydrogen or an L1 linker covalently bound to Ab and D;
L1-Y is hydrogen or an L1 linker covalently bound to Ab and D;
q is 1 or 0;
and ,
p has a value of about 1 to about 8.
2. The conjugate according to example 1, wherein R 3 Is cyano.
3. The conjugate according to example 1, wherein R 3 Is that
Figure BDA0004113777320000892
4. The conjugate according to example 1, wherein R 3 Is that
Figure BDA0004113777320000893
5. The conjugate according to example 1, wherein R 1A 、R 1B and R1C Each independently is hydrogen orMethyl group.
6. The conjugate according to example 5, wherein R 1A and R1B Each methyl.
7. The conjugate of example 6, wherein E3LB has the formula:
Figure BDA0004113777320000901
/>
Figure BDA0004113777320000911
8. the conjugate of embodiment 1, wherein in each instance, L1 is independently a linker selected from the group consisting of:
Figure BDA0004113777320000912
Figure BDA0004113777320000921
wherein ,
j is-CH 2 -CH 2 -CH 2 -NH-C(O)-NH 2 ；—CH 2 -CH 2 -CH 2 -CH 2 -NH 2 ；—CH 2 -CH 2 -CH 2 -CH 2 -NH-CH 3 The method comprises the steps of carrying out a first treatment on the surface of the or-CH 2 -CH 2 -CH 2 -CH 2 -N(CH 3 ) 2 ；
R 5 and R6 Independently hydrogen or C 1-5 An alkyl group; or R is 5 and R6 Together with the nitrogen to which each is attached, form an optionally substituted 5-to 7-membered heterocyclyl;
R 7 and R8 Each independently is hydrogen, halo, C 1-5 Alkyl, C 1-5 Alkoxy or hydroxy;
Figure BDA0004113777320000922
and wherein
Figure BDA0004113777320000923
Is the point of attachment to Ab.
9. The conjugate of example 8, having the following structure:
Figure BDA0004113777320000931
10. the conjugate according to embodiment 1, wherein L1-T is a linker.
11. The conjugate of embodiment 1, wherein L1-U or L1-V is a linker.
12. The conjugate of embodiment 1, wherein L1-Y is a linker and q is 1.
Figure BDA0004113777320000932
13. The conjugate according to example 12, wherein L1-Y has the structure,
Figure BDA0004113777320000933
14. the conjugate according to example 1, wherein
L1-T is a linker;
L1-U and L1-T are each hydrogen; and is also provided with
q is zero.
15. The conjugate of embodiment 1, wherein z is zero.
16. The conjugate of embodiment 1, wherein z is one.
17. The conjugate according to example 1, wherein R 2 Is hydrogen, methyl, ethyl or propyl.
18. The conjugate according to example 17, wherein R 2 Is methyl.
19. The conjugate according to example 18, wherein R 2 Combined with E3LB as
Figure BDA0004113777320000941
20. The conjugate according to example 1, wherein Y 1 and Y2 Each is-CH.
21. The conjugate according to example 1, wherein Y 1 Is N and Y 2 is-CH.
22. The conjugate according to example 1, wherein Y 1 is-CH and Y 2 Is N.
23. The conjugate according to example 1, wherein R 4 Is hydrogen.
24. The conjugate according to example 1, wherein R 4 Is methyl.
25. The conjugate according to example 24, wherein R 4 Methyl is shown below:
Figure BDA0004113777320000942
26. the conjugate of embodiment 1, wherein Ab is an antibody that binds to one or more polypeptides selected from the group consisting of CD71, trop2, naPi2b, ly6E, epCAM, MSLN, and CD 22.
27. The conjugate of embodiment 26, wherein Ab is an antibody that binds to one or more polypeptides selected from the group consisting of CD71 and Trop 2.
28. The conjugate of example 1, wherein PB is a protein binding group covalently bound to L2, having the structure:
Figure BDA0004113777320000951
29. the conjugate according to example 1, having formula Ia:
Figure BDA0004113777320000952
wherein ,
L1-T is a linker covalently bound to Ab;
ab is an antibody that binds to one or more polypeptides selected from the group consisting of CD71, trop2, naPi2b, ly6E, epCAM, MSLN, and CD 22.
PB is a protein binding group covalently bound to L2, which has the following structure:
Figure BDA0004113777320000961
/>
l2 is selected from the group consisting of L2a, L2b, and L2 c;
and ,
the value of p is from about 4 to about 8.
30. The conjugate of embodiment 29, wherein L1-T is a linker selected from the group consisting of:
Figure BDA0004113777320000962
Figure BDA0004113777320000971
wherein ,
Figure BDA0004113777320000972
is the point of attachment to Ab. />
31. The conjugate according to embodiment 29, wherein L2 is L2a.
32. The conjugate according to embodiment 31, wherein G is
Figure BDA0004113777320000981
33. The conjugate according to example 31, wherein R 4 Is methyl.
34. The conjugate of embodiment 29, wherein PB is:
Figure BDA0004113777320000982
35. the conjugate of embodiment 29, wherein p has a value of about 5 to about 7.
36. The conjugate of example 1, having the following structure:
Figure BDA0004113777320000983
/>
Figure BDA0004113777320000991
37. the conjugate of example 1, having the following structure:
Figure BDA0004113777320000992
/>
Figure BDA0004113777320001001
38. a pharmaceutical composition comprising the conjugate of example 1 and one or more pharmaceutically acceptable excipients.
39. A method of treating a disease in a human in need thereof, the method comprising administering to the human an effective amount of the conjugate of example 1 or the composition of example 38.
40. The method of embodiment 39, wherein the disease is cancer.
41. The method of embodiment 40, wherein the cancer is BRM-dependent.
42. The method of embodiment 40, wherein the cancer is non-small cell lung cancer.
43. A method of reducing target BRM protein levels in a subject, comprising:
administering to the subject the conjugate of example 1 or the composition of example 38, wherein the PB moiety binds to the target BRM protein, wherein ubiquitin ligase affects degradation of the bound target BRM protein, wherein the level of the BRM target protein is reduced.
IV. preparation
Pharmaceutical formulations of therapeutic Ab-CIDE as described herein may be prepared for parenteral administration, e.g., with a pharmaceutically acceptable parenteral carrier and may be injected in unit dose injection form, intravenously, intratumorally. Ab-CIDE of desired purity is optionally admixed with one or more pharmaceutically acceptable excipients (Remington's Pharmaceutical Sciences (1980) 16 th edition, osol, main code) in the form of a lyophilized formulation for reconstitution or in the form of an aqueous solution.
Ab-CIDE can be formulated into pharmaceutical compositions according to standard pharmaceutical practice. According to this aspect, there is provided a pharmaceutical composition comprising Ab-CIDE in combination with one or more pharmaceutically acceptable excipients.
Typical formulations are prepared by mixing the Ab-CIDE with excipients such as carriers and/or diluents. Suitable carriers, diluents and other excipients are well known to those skilled in the art and include materials such as carbohydrates, waxes, water soluble and/or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like. The particular carrier, diluent or other excipient used will depend on the manner and purpose in which the Ab-CIDE is to be administered. Solvents are generally selected based on the recognition by those skilled in the art that safe solvents (GRAS) are administered to mammals.
Generally, the safe solvent is a non-toxic aqueous solvent, such as water and other non-toxic solvents that are soluble in or miscible with water. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycols (e.g., PEG 400, PEG 300), and the like, and mixtures thereof. Acceptable diluents, carriers, excipients and stabilizers are non-toxic to the recipient at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and others An organic acid; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethyldiammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl p-hydroxybenzoates such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); a low molecular weight (less than about 10 residues) polypeptide; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc protein complexes); and/or nonionic surfactants, e.g. TWEEN TM 、PLURONICS TM Or polyethylene glycol (PEG).
The formulation may also contain one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, flavoring agents and other known additives to provide an aesthetically pleasing Ab-CIDE display or to aid in the preparation of a pharmaceutical product. Conventional dissolution and mixing procedures can be used to prepare the formulations.
Formulation may be carried out by mixing with a physiologically acceptable carrier (i.e., a carrier that is non-toxic to the receptor at the dosage and concentration employed) at an appropriate pH and desired purity at ambient temperature. The pH of the formulation will depend primarily on the particular use and concentration of the compound, but may be in the range of about 3 to about 8. Formulations in acetate buffer at pH 5 are suitable examples.
Ab-CIDE formulations may be sterile. In particular, the formulation to be used for in vivo administration must be sterile. Such sterilization is readily accomplished by filtration through sterile filtration membranes.
Ab-CIDE can generally be stored as a solid composition, a lyophilized formulation, or as an aqueous solution.
Pharmaceutical compositions comprising Ab-CIDE can be formulated, administered and administered in a manner consistent with good medical practice, i.e., dosage, concentration, schedule, course of treatment, vehicle and route of administration. Factors to be considered in this case include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to the practitioner. The "therapeutically effective amount" of the compound to be administered will be constrained by such considerations and is the minimum amount required to prevent, ameliorate or treat coagulation factor mediated disorders. The amount is preferably less than an amount that is toxic to the host or that renders the host significantly more prone to bleeding.
Ab-CIDE can be formulated into pharmaceutical dosage forms to provide easily controlled drug dosages and to enable patients to follow prescriptions. Depending on the method used to administer the drug, the pharmaceutical composition (or formulation) for administration may be packaged in a variety of ways. Typically, the articles for dispensing include containers in which the pharmaceutical formulation is deposited in a suitable form. Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass), sachets, ampoules, plastic bags, metal cylinders, and the like. The container may also include an tamper-proof assembly to prevent inadvertent access to the contents of the package. In addition, the container is provided with a label describing the contents of the container. Appropriate warnings may also be included on the label.
The pharmaceutical composition may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. The suspensions may be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, such as 1, 3-butanediol. The sterile injectable preparation may also be prepared as a lyophilized powder. Acceptable vehicles and solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
The amount of Ab-CIDE that can be combined with a carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a time release formulation for oral administration to humans may contain from about 1 to 1000mg of the active agent formulated with a suitable and convenient amount of carrier material, which may constitute from about 5% to about 95% (weight: weight) of the total composition. The pharmaceutical compositions may be prepared to provide an easily measurable dosage. For example, an aqueous solution for intravenous infusion may contain about 3 μg to 500 μg of active ingredient per milliliter of solution so that a suitable volume may be infused at a rate of about 30 mL/h.
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes so as to render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
The formulations may be packaged in unit-dose or multi-dose containers, for example sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, for injection prior to use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose, or an appropriate fraction thereof, of an active ingredient as described herein above.
The subject of the present invention is further a veterinary composition comprising at least one active ingredient as defined above and a veterinary carrier. Veterinary carriers are materials useful for administering the compositions, and may be solid, liquid or gaseous materials that are inert or acceptable in the veterinary arts and are compatible with the active ingredient. These veterinary compositions may be administered parenterally or by any other desired route.
V. indications and methods of treatment
The Ab-CIDE disclosed herein is expected to be useful in the treatment of various diseases or conditions associated with BRM. Also provided herein are Ab-CIDE or compositions comprising Ab-CIDE for use in therapy. In some embodiments, provided herein are Ab-CIDE or compositions comprising Ab-CIDE for use in treating or preventing diseases and disorders disclosed herein. Also provided herein is the use of Ab-CIDE or a composition comprising Ab-CIDE in therapy. In some embodiments, provided herein is the use of Ab-CIDE for treating or preventing diseases and disorders disclosed herein. Also provided herein is the use of Ab-CIDE or a composition comprising Ab-CIDE in the manufacture of a medicament for the treatment or prevention of the diseases and conditions disclosed herein.
Typically, the disease or disorder to be treated is a BRM-dependent disease or disorder, for example a hyperproliferative disease (such as cancer). Examples of cancers to be treated herein include BRM-dependent cancers. In certain embodiments, the cancer is non-small cell lung cancer.
In certain embodiments, the subject matter described herein relates to a method of reducing the level of a target BRM protein in a subject, the method comprising:
administering to a subject an Ab-CIDE as described herein or a composition comprising an Ab-CIDE as described herein, wherein the PB moiety binds to a target BRM protein, wherein ubiquitin ligase affects degradation of the bound target BRM protein, wherein the level of the BRM target protein is reduced.
In certain embodiments, ab-CIDE comprising anti-NaPi 2b antibodies (e.g., those described above) is used in a method of treating a solid tumor (e.g., an ovary). In certain embodiments, ab-CIDE comprising an anti-CD 71, trop2, naPi2b, ly6E, epCAM, MSLN or CD22 antibody is used in a method of treating a tumor or cancer.
Ab-CIDE can be administered by any route suitable for the condition to be treated. Ab-CIDE is typically administered parenterally, i.e., by infusion, subcutaneously, intramuscularly, intravenously, intradermally, intrathecally, and epidurally.
Ab-CIDE can be used alone or in combination with other agents in therapy. For example, ab-CIDE can be co-administered with at least one additional therapeutic agent. Such combination therapies as described above encompass both co-administration (wherein two or more therapeutic agents are contained in the same composition or separate formulations) and separate administration, in which case the administration of the Ab-CIDE may occur before, simultaneously with and/or after administration of additional therapeutic agents and/or adjuvants. Ab-CIDE may also be used in combination with radiation therapy.
Ab-CIDE (and any additional therapeutic agents) may be administered by any suitable means, including parenteral, intrapulmonary and intranasal administration, and if topical treatment is desired, intralesional administration may be employed. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including but not limited to single or multiple administrations at various points in time, bolus administrations, and pulse infusion.
For the prevention or treatment of disease, the appropriate dosage of Ab-CIDE (when used alone or in combination with one or more other drugs) will depend on the type of disease to be treated, the type of Ab-CIDE, the severity and course of the disease, whether Ab-CIDE is administered for prophylactic or therapeutic purposes, previous treatments, patient history and response to Ab-CIDE, as appropriate to the attending physician. Ab-CIDE is suitably administered to a patient at one time or over a series of treatments. Depending on the type and severity of the disease, an Ab-CIDE of about 1 μg/kg to 15mg/kg (e.g., 0.1 mg/kg-10 mg/kg) may be the initial candidate dose administered to the patient, for example, by one or more separate administrations or by continuous infusion. Depending on the factors mentioned above, a typical daily dose may range from about 1 μg/kg to 100mg/kg or more. For repeated administrations over several days or longer, depending on the condition, the treatment will generally continue until the desired inhibition of disease symptoms occurs. An exemplary dosage of Ab-CIDE will be in the range of about 0.05mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg, or 10mg/kg (or any combination thereof) may be administered to a patient. Such doses may be administered intermittently, e.g., weekly or every three weeks (e.g., such that the patient receives about two to about twenty, or e.g., about six doses). An initial higher loading dose may be administered followed by one or more lower doses. However, other dosage regimens may be useful. The progress of this therapy is readily monitored by conventional techniques and assays.
The methods described herein include methods of degrading a target protein. In certain embodiments, the method comprises administering Ab-CIDE to a subject, wherein the target protein is degraded. The degradation level of the protein may be about 1% to about 5%; or about 1% to about 10%; or about 1% to about 15%; or about 1% to about 20%; or about 1% to about 30%; or about 1% to about 40%; about 1% to about 50%; or about 10% to about 20%; or about 10% to about 30%; or about 10% to about 40%; or about 10% to about 50%; or at least about 1%; or at least about 10%; or at least about 20%; or at least about 30%; or at least about 40%; or at least about 50%; or at least about 60%; or at least about 70%; or at least about 80%; or at least about 90%; or at least about 95%; or at least about 99%.
Methods described herein include methods of reducing proliferation of tumor tissue (such as non-small cell lung cancer). In certain embodiments, the methods comprise administering Ab-CIDE to a subject, wherein proliferation of tumor tissue is reduced. The level of reduction may be about 1% to about 5%; or about 1% to about 10%; or about 1% to about 15%; or about 1% to about 20%; or about 1% to about 30%; or about 1% to about 40%; about 1% to about 50%; or about 10% to about 20%; or about 10% to about 30%; or about 10% to about 40%; or about 10% to about 50%; or at least about 1%; or at least about 10%; or at least about 20%; or at least about 30%; or at least about 40%; or at least about 50%; or at least about 60%; or at least about 70%; or at least about 80%; or at least about 90%; or at least about 95%; or at least about 99%.
VI. products
In another aspect, described herein is an article of manufacture (e.g., a kit) containing materials for treating the above-described diseases and conditions. The kit comprises a container comprising Ab-CIDE. The kit may further comprise a label or package insert on or associated with the container. The term "package insert" is used to refer to instructions generally included in commercial packages of therapeutic products that contain information concerning the indication, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
Suitable containers include, for example, bottles, vials, syringes, blister packs, and the like. A "vial" is a container suitable for holding a liquid or lyophilized formulation. In one embodiment, the vial is a disposable vial, such as a 20cc disposable vial with a stopper. The container may be formed from a variety of materials such as glass or plastic. The container may contain an Ab-CIDE or formulation thereof that is effective for the treatment of the condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle).
At least one active agent in the composition is Ab-CIDE. The label or package insert indicates that the compound is used to treat a selected disorder, such as cancer. Additionally, the label or package insert may indicate that the patient to be treated is a patient suffering from a condition such as a hyperproliferative condition, atherosclerosis, neurodegeneration, cardiac hypertrophy, pain, migraine or neurotraumatic disease or event. In one embodiment, the label or package insert indicates that a composition comprising Ab-CIDE can be used to treat a disorder caused by abnormal cell growth. The label or package insert may also indicate that the composition may be used to treat other conditions. Alternatively or additionally, the article of manufacture may further comprise a second container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. The kit may further include other substances, including other buffers, diluents, filters, needles and syringes, as desired from a commercial and user perspective.
The kit may further comprise an administration guidance for the Ab-CIDE and the second pharmaceutical formulation (if present). For example, if the kit comprises a first composition comprising Ab-CIDE and a second pharmaceutical formulation, the kit may further comprise instructions for simultaneous, sequential or separate administration of the first pharmaceutical composition and the second pharmaceutical composition to a patient in need thereof.
In another embodiment, the kit is suitable for delivering Ab-CIDE in solid oral form, such as a tablet or capsule. Such kits preferably comprise a plurality of unit doses. Such kits may include cards with doses oriented in the order of their intended use. An example of such a kit is a blister pack. Blister packages are well known in the packaging industry and are widely used for packaging pharmaceutical unit dosage forms. If desired, memory assistance may be provided, for example, in the form of numbers, letters, or other indicia, or with calendar insertion events specifying the dates on which doses may be administered in the treatment schedule.
According to one embodiment, a kit may comprise (a) a first container having Ab-CIDE contained therein; and optionally (b) a second container having a second pharmaceutical formulation contained therein, wherein the second pharmaceutical formulation comprises a second compound having anti-hyperproliferative activity. Alternatively or additionally, the kit may further comprise a third container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. The kit may further include other substances, including other buffers, diluents, filters, needles and syringes, as desired from a commercial and user perspective.
In certain other embodiments, wherein the kit comprises Ab-CIDE and a second therapeutic agent, the kit may comprise containers for holding separate compositions (e.g., separate bottles or separate foil packages), however, the separate compositions may also be held in a single, non-separate container. Typically, the kit includes instructions for administering the individual components. The kit form is particularly advantageous when the individual components are preferably administered in different dosage forms (e.g., oral and parenteral), at different dosage intervals, or when the prescribing physician wishes to titrate the various components of the combination.
Methods of preparing conjugates
Synthetic route
The subject matter described herein also relates to methods of preparing CIDE, L1-CIDE and Ab-CIDE from L1-CIDE. Generally, the method comprises contacting an antibody, or variant, mutation, splice variant, insertion/deletion, and fusion thereof, with L1-CIDE under conditions wherein the antibody is covalently bound to any available point of attachment on the L1-CIDE, wherein Ab-CIDE is prepared. The subject matter described herein also relates to methods of making Ab-CIDE from Ab-L1 moieties (i.e., antibodies, or variants, mutations, splice variants, insertions/deletions, and fusions thereof, covalently linked to L1), comprising contacting CIDE with Ab-L1 under conditions wherein CIDE is covalently bound to any available point of attachment on Ab-L1, wherein Ab-CIDE is made. The method may further comprise conventional isolation and purification of Ab-CIDE.
CIDE, L1-CIDE and Ab-CIDE and other compounds described herein may be synthesized by synthetic routes including, particularly in view of the description contained herein, processes similar to those known in the chemical arts, and those described below for other heterocycles: comprehensive Heterocyclic Chemistry II, katritzky and Rees master, elsevier,1997, e.g. volume 3; liebigs Annalen der Chemie, (9): 1910-16, (1985); helvetica Chimica Acta,41:1052-60, (1958); arzneimittel-Forschung,40 (12): 1328-31, (1990). The starting materials are generally available from commercial sources, such as Aldrich Chemicals (Milwaukee, WI), or are readily prepared using methods well known to those skilled in the art (e.g., by methods generally described by Louis F. Fieser and Mary Fieser, reagents for Organic Synthesis, v.1-23, wiley, N.Y. (1967-2006 Main) or Beilsteins Handbuch der organischen Chemie,4, aufl. Edit Springer-Verlag, berlin, including journals (also obtained by the Beilstein on-line database)).
Synthetic chemical transformations and protecting group methods (protection and deprotection) and the necessary reagents and intermediates useful for synthesizing CIDE, L1-CIDE and Ab-CIDE and other compounds described herein are known in the art and include, for example, those described in the following documents: larock, comprehensive Organic Transformations, VCH Publishers (1989); T.W.Greene and P.G.M.Wuts, protective Groups in Organic Synthesis, 3 rd edition, john Wiley and Sons (1999); paquette edit, encyclopedia of Reagents for Organic Synthesis, john Wiley and Sons (1995) and subsequent versions thereof. In the preparation of CIDE, L1-CIDE and Ab-CIDE and other compounds, it may be desirable to protect the distal functional groups (e.g., primary or secondary amines) of the intermediates. The need for such protection will vary depending on the nature of the distal functionality and the conditions of the preparation process. Suitable amino protecting groups include acetyl, trifluoroacetyl, t-Butoxycarbonyl (BOC), benzyloxycarbonyl (CBz or CBZ) and 9-fluorenylmethyleneoxycarbonyl (Fmoc). Whether such protection is required is readily determinable by one of skill in the art. For a general description of protecting groups and their use, see T.W. Greene, protective Groups in Organic Synthesis, john Wiley & Sons, new York,1991.
General procedures and examples provide exemplary methods for preparing CIDE, L1-CIDE and Ab-CIDE as well as other compounds described herein. Those skilled in the art will appreciate that other synthetic routes may be used to synthesize Ab-CIDE and compounds. Although specific starting materials and reagents are described and discussed in the schemes, general procedures, examples, other starting materials and reagents may be readily substituted to provide various derivatives and/or reaction conditions. Furthermore, in accordance with the present disclosure, many of the exemplary compounds prepared by the methods may be further modified using conventional chemical methods well known to those skilled in the art.
In general, ab-CIDE can be prepared by linking CIDE with L1 linker to prepare L1-CIDE and conjugating L1-CIDE with any of the antibodies described herein or variants, mutations, splice variants, insertions/deletions and fusions thereof (including cysteine engineered antibodies) according to the procedures of WO 2013/055987, WO 2015/023555, WO 2010/009124, WO 2015/095227. Alternatively, ab-CIDE may be prepared by first linking an antibody or variant, mutation, splice variant, insertion/deletion, and fusion described herein (including cysteine engineered antibodies) with an L1 linker and conjugating it to any CIDE.
The following synthetic schemes describe exemplary methods for preparing CIDE, L1-CIDE and Ab-CIDE, as well as other compounds and components thereof. Other synthetic routes for preparing CIDE, L1-CIDE and Ab-CIDE, as well as other compounds and components thereof, are disclosed elsewhere herein.
1. Joint L1
With respect to linker L1, schemes 1-4 depict synthetic routes to exemplary linker L1 linked to antibody Ab via disulfide. Ab is linked to L1 by disulfide bonds, while CIDE is linked to L1 by any available point of attachment on CIDE.
Figure BDA0004113777320001091
Referring to scheme 1, 2-bis (pyridin-2-yl) disulfane and 2-mercaptoethanol are reacted in pyridine and methanol at room temperature to give 2- (pyridin-2-yl-disulfanyl) ethanol. Acylation with 4-nitrophenyl chloroformate in triethylamine and acetonitrile gives 4-nitrophenyl 2- (pyridin-2-yldisulfanyl) ethylcarbonate 9.
Figure BDA0004113777320001092
Referring to scheme 2, HOAc (0.1 mL) was added to a mixture of 1, 2-bis (5-nitropyridin-2-yl) disulfane 10 (1.0 g,3.22 mmol) in anhydrous DMF/MeOH (25 mL/25 mL) followed by 2-aminoethylthiol hydrochloride 11 (183 mg,1.61 mmol). The reaction mixture was stirred at room temperature overnight, concentrated in vacuo to remove solvent, and the residue was washed with DCM (30 ml×4) to give 2- ((5-nitropyridin-2-yl) disulfanyl) ethanamine hydrochloride 12 (300 mg, 69.6%) as a pale yellow solid. 1 H NMR(400MHz,DMSO-d 6 )δ9.28(d,J＝2.4Hz,1H),8.56(dd,J＝8.8,2.4Hz,1H),8.24(s,4H),8.03(d,J＝8.8Hz,1H),3.15-3.13(m,2H),3.08-3.06(m,2H)。
Figure BDA0004113777320001101
Referring to scheme 3, at N 2 Next, 1, 2-bis (5-nitropyridin-2-yl) disulfane 10 (9.6 g,30.97 mmol) and 2-mercaptoethanol (1.21 g,15.49 mmol) were added to the mixture in dry DCM/CH 3 The OH (250 mL/250 mL) solution was stirred at room temperature for 24 hours. After concentrating the mixture under vacuumThe residue was diluted with DCM (300 mL). Adding MnO 2 (10g) And the mixture was stirred at room temperature for an additional 0.5h. The mixture was purified by silica gel column chromatography (DCM/meoh=100/1 to 100/1) to give 2- ((5-nitropyridin-2-yl) disulfanyl) ethanol 13 (2.2 g, 61.1%) as a brown oil. 1 HNMR(400MHz,CDCl 3 )δ9.33(d,J＝2.8Hz,1H),8.38-8.35(dd,J＝9.2,2.8Hz,1H),7.67(d,J＝9.2Hz,1H),4.10(t,J＝7.2Hz,1H),3.81-3.76(q,2H),3.01(t,J＝5.2Hz,2H)。
To a solution of 13 (500 mg,2.15 mmol) in anhydrous DMF (10 mL) was added DIEA (404 mg,6.45 mmol) followed by PNP carbonate (bis (4-nitrophenyl) carbonate, 1.31g,4.31 mmol). The reaction solution was stirred at room temperature for 4 hours, and the mixture was purified by preparative HPLC (FA) to give 4-nitrophenyl 2- ((5-nitropyridin-2-yl) disulfanyl) ethylcarbonate 14 (270 mg, 33.1%) as a light brown oil. 1 H NMR(400MHz,CDCl 3 )δ9.30(d,J＝2.4Hz,1H),8.43-8.40(dd,J＝8.8,2.4Hz,1H),8.30-8.28(m,2H),7.87(d,J＝8.8Hz,1H),7.39-7.37(m,2H),4.56(t,J＝6.4Hz,2H),3.21(t,J＝6.4Hz,2H)。
Figure BDA0004113777320001111
Referring to scheme 4, sulfuryl chloride (2.35 mL,1.0M in DCM, 2.35 mmol) was added dropwise to a stirred suspension of 5-nitropyridine-2-thiol (334 mg,2.14 mmol) in dry DCM (7.5 mL) under 0deg.C (ice/acetone) and argon atmosphere. The reaction mixture was changed from yellow suspension to yellow solution, allowed to warm to room temperature, then stirred for 2 hours, and then the solvent was removed by vacuum evaporation to give a yellow solid. The solid was redissolved in DCM (15 mL) and treated dropwise with a solution of (R) -2-mercaptopropan-1-ol (213 mg,2.31 mmol) in dry DCM (7.5 mL) at 0deg.C under an argon atmosphere. The reaction mixture was allowed to warm to room temperature and stirred for 20 hours, at which time analysis by LC/MS showed formation of a large amount of product (es+) M/z 247 ([ m+h) at a retention time of 1.41 minutes ] +. About 100% relative intensity). The precipitate was removed by filtration and the filtrate evaporated in vacuo to give an orange solid which was taken up in H 2 O (20 mL) and basified with ammonium hydroxide solution. The mixture was extracted with DCM (3X 25 mL) and the combined extracts were taken up in H 2 O (20 mL), brine (20 mL), and dried (MgSO 4 ) Filtration and evaporation in vacuo gave the crude product. Purification by flash chromatography (gradient elution in 1% increments: 100% DCM to 98:2v/v DCM/MeOH) afforded (R) -2- ((5-nitropyridin-2-yl) disulfanyl) propan-1-ol 15 as an oil (111 mg, 21% yield).
At 20 ℃, to triphosgene solution, i.e. Cl-containing 3 COCOOCCl 3 Sigma Aldrich, CAS Reg.No.32315-10-9 (241 mg,0.812 mmol) in DCM (10 mL) was added dropwise a solution of (R) -2- ((5-nitropyridin-2-yl) disulfanyl) propan-1-ol 15 (500 mg,2.03 mmol) and pyridine (153 mg,1.93 mmol) in DCM (10 mL). After stirring the reaction mixture at 20 ℃ for 30min, it is concentrated and (R) -2- ((5-nitropyridin-2-yl) disulfanyl) propyl chloroformate 16 is used directly without further purification to covalently link to any available group on the CIDE through a chloroformate group.
2. Cysteine engineered antibodies
For cysteine engineered antibodies conjugated by reduction and reoxidation, they can generally be prepared as follows. The light chain amino acids are numbered according to Kabat (Kabat et al, sequences of proteins of immunological interest, (1991) 5 th edition, US Dept of Health and Human Service, national Institutes of Health, bethesda, md.). The heavy chain amino acids are numbered according to the EU numbering system (Edelman et al (1969) Proc. Natl. Acad. Of Sci.63 (1): 78-85), except for the Kabat system. Single letter amino acid abbreviations are used.
Full length cysteine engineered monoclonal antibodies (THIOMAB) expressed in CHO cells TM Antibodies) with cysteine adducts (cystine) or are glutathionylated on engineered cysteines due to cell culture conditions. Thus, thiomab purified from CHO cells TM The antibody cannot be conjugated to a Cys reactive linker L1-CIDE intermediate. By use of reducing agents such as DTT (Cleland reagent, dithiothreitol) or TCEP (tris (2-carboxyethyl) phosphine hydrochloride; getz et al (1999) Anal biochem Vol 273:73-80;Soltec Ventures,Beve)rly, MA), followed by the reformation of interchain disulfide bonds (reoxidation) with a mild oxidizing agent such as dehydroascorbic acid, can be used to conjugate the cysteine engineered antibodies with the L1-CIDE intermediates described herein. Full length cysteine engineered monoclonal antibodies (THIOMAB) to be expressed in CHO cells TM Antibodies) (Gomez et al (2010) Biotechnology and bioeng.105 (4): 748-760; gomez et al (2010) Biotechnol. Prog. 26:1438-1445) reduced, for example, with about 50-fold excess of DTT overnight at room temperature in 50mM Tris, pH 8.0 and 2mM EDTA, which can remove Cys and glutathione adducts and reduce inter-chain disulfide bonds of antibodies. Removal of the adduct was monitored by reverse phase LCMS using PLRP-S chromatography column. By reduction of THIOMAB TM The antibodies were added to at least four volumes of 10mM sodium succinate, diluted and acidified with pH 5 buffer.
Alternatively, the antibody is diluted and acidified by adding the antibody to at least four volumes of 10mM succinate, pH 5 buffer, and titrating with 10% acetic acid until the pH is about five. Subsequent pH-reduced and diluted THIOMAB TM The antibody was loaded onto a HiTrap S cation exchange column, washed with several column volumes of 10mM sodium acetate, pH 5, and eluted with 50mM Tris, pH8.0, 150mM sodium chloride. By performing reoxidation, disulfide bonds are reestablished between cysteine residues present in the parent Mab. The eluted reduced THIOMAB TM The antibodies were treated with 15X dehydroascorbic acid (DHAA) for about 3 hours at room temperature, or alternatively, 200nM to 2mM copper sulfate in water (CuSO 4 ) Treatment was carried out overnight. Other oxidizing agents (i.e., oxidizing agents) and oxidizing conditions known in the art may be used. Ambient air oxidation may also be effective. This mild partial reoxidation step can effectively form intrachain disulfide bonds with high fidelity. Reoxidation was monitored by reverse phase LCMS using PLRP-S chromatography column. As described above, the thioMAB to be reoxidized TM The antibodies were diluted to a pH of about 5 with succinate buffer and purified on S column as described above, except that gradient elution was performed: 10mM succinate, pH 5, 300mM sodium chloride (buffer B), 10mM succinate, pH 5 solution (buffer A). Thiomab eluted to TM Antibodies toEDTA was added to a final concentration of 2mM and, if necessary, concentrated to a final concentration exceeding 5mg/mL. The resulting THIOMAB will be ready for conjugation TM Antibody aliquots were stored at-20℃or-80 ℃. Liquid chromatography/mass spectrometry was performed on a 6200 series TOF or QTOF Agilent LC/MS. PRLP-heating the sample to 80 ℃
Figure BDA0004113777320001131
1000 A microporous chromatographic column (50 mm. Times. 2.1mm,Polymer Laboratories,Shropshire,UK) was used for chromatographic separation. The eluate was ionized directly using an electrospray source using a linear gradient of 30-40% B (solvent A: 0.05% TFA in water and solvent B: 0.04% TFA in acetonitrile). Data were collected and deconvolved using MassHunter software (Agilent). Prior to LC/MS analysis, the antibody or conjugate (50. Mu.g) was treated with PNGase F (2 units/ml; PROzyme, san Leandro, calif.) at 37℃for 2 hours to remove the N-linked carbohydrate.
Alternatively, the antibody or conjugate was partially digested with LysC (0.25 μg/50 μg (microgram) antibody or conjugate) at 37 ℃ for 15 minutes to give Fab and Fc fragments for LCMS analysis. Peaks in the deconvolved LCMS spectra were assigned and quantified. The ratio of CIDE to antibody (CAR) is calculated by calculating the intensity ratio of one or more peaks corresponding to the CIDE-conjugated antibody relative to all peaks observed.
3. Conjugation of linker L1-CIDE groups to antibodies
In one method of conjugating linker L1-CIDE compounds to antibodies, after the above reduction and reoxidation steps, cysteine-containing engineered antibodies (thioMAB) are conjugated with 1M Tris TM Antibodies), pH 5, 150mM NaCl,2mM EDTA to pH7.5-8.5. An excess (about 3 moles to 20 equivalents) of linker-CIDE intermediate having thiol-reactive groups (e.g., maleimide or 4-nitropyridine disulfide or Methane Thiosulfonyl (MTS) disulfide) is dissolved in DMF, DMA or propylene glycol and added to the reduced, reoxidized and pH adjusted antibody. The reaction is incubated at room temperature or 37C and monitored until completion (1 to about 24 hours), e.g., by LC-MS partitioning of the reaction mixtureAnd (5) analyzing and determining. After the reaction is complete, the conjugate may be purified by one or any combination of methods in order to remove residual unreacted L1-CIDE intermediates and aggregate proteins (if present at high levels). For example, the conjugate may be diluted with 10mM histidine acetate (pH 5.5) until a final pH of about 5.5, and then purified by S cation exchange chromatography using a HiTrap S column or S maxi spin column (Pierce) coupled to an Akta purification system (GE Healthcare). Alternatively, the conjugate may be purified by gel filtration chromatography using an S200 column or Zeba centrifugation column connected to an Akta purification system. Alternatively, dialysis may be used. Thiomab using gel filtration or dialysis TM The antibody CIDE conjugate was formulated into 20mM His/acetate (pH 5), 240mM sucrose. The purified conjugate was concentrated by centrifugal ultrafiltration and filtered through a 0.2 μm filter under sterile conditions, followed by cryopreservation. Ab-CIDE was characterized by BCA assay to determine protein concentration, SEC (size exclusion chromatography) was analyzed for aggregation analysis, and LC-MS was performed after treatment with lysine C endopeptidase (LysC) to calculate CAR.
Size exclusion chromatography of the conjugates was performed using a Shodex KW802.5 column with 0.2M potassium phosphate (pH 6.2) with 0.25mM potassium chloride and 15% IPA at a flow rate of 0.75 ml/min. The aggregation state of the conjugate was determined by integrating the peak area absorbance eluting at 280 nm.
LC-MS analysis of Ab-CIDE can be performed using an Agilent QTOF 6520ESI instrument. For example, the CAR was treated with Tris (pH 7.5) containing 1:500w/w intracellular protease Lys C (Promega) for 30min at 37 ℃. Loading the resulting cleaved fragments to a temperature of 80℃C
Figure BDA0004113777320001141
(angstrom), 8 μm (micrometer) PLRP-S (highly crosslinked polystyrene) column and eluted with a 30% B to 40% B gradient over 5 minutes. Mobile phase a was 0.05% TFA H 2 O solution, and mobile phase B was 0.04% TFA in acetonitrile. The flow rate was 0.5ml/min. Protein elution was monitored by UV absorbance detection at 280nm prior to electrospray ionization and MS analysis. Unconjugated Fc fragments, residues, can generally be achieved Chromatographic resolution of unconjugated Fab and pharmaceutical Fab. Using Mass Hunter TM Software (Agilent Technologies) deconvolves the obtained m/z spectra to calculate the mass of the antibody fragments.
General synthetic method
The preparation of Ab (L1-D) having the chemical structure is described below p General procedure for conjugates of (2).
1.1 general synthetic methods for coupling L2 with E3LB to prepare E3LB-L2 intermediates
In certain embodiments, L2 is first contacted with a first suitable solvent, a first base, and a first coupling agent to produce a first solution. In certain embodiments, L2 is contacted with the first suitable solvent, the first base, and the first coupling agent at room temperature (about 25 ℃) for about 15 minutes. E3LB was then contacted with the first solution.
In certain embodiments, E3LB is contacted with the first solution for about one hour at room temperature (about 25 ℃). The solution is then concentrated and optionally purified.
In certain embodiments, the molar ratio of L2 to first base to first coupling agent is about 1:4:1.19. In certain embodiments, the molar ratio of L2 to first base to first coupling agent is about 1:2:0.5, about 1:3:1, about 1:4:2, about 1:5:3, or about 1:6:4.
In certain embodiments, the molar ratio of L2 to E3LB is about 1:1. In certain embodiments, the molar ratio of L2 to E3LB is about 1:0.5, about 1:0.75, about 1:2, or about 0.5:1.
1.2 general synthetic methods for coupling E3LB-L2 intermediates with PB to prepare CIDE
In certain embodiments, the E3LB-L2 intermediate is coupled to PB to make CIDE. In certain embodiments, the PB is first contacted with a second suitable solvent, a second base, and a second coupling agent. In certain embodiments, the contacting is performed at room temperature (about 25 ℃) for about 10 minutes. The solution was then contacted with E3LB-L2 intermediate. In certain embodiments, the second solution is contacted with the E3LB-L2 intermediate at room temperature (about 25 ℃) for about 1 hour. The solution is then concentrated and optionally purified to prepare CIDE.
In certain embodiments, the molar ratio of PB to second base to second coupling agent is about 1:4:1.2. In certain embodiments, the molar ratio of PB to second base to second coupling agent is about 1:3:0.75, about 1:5:1, about 1:3:2, or about 1:5:3.
In certain embodiments, the molar ratio of PB to E3LB-L2 intermediate is about 1:1. In certain embodiments, the molar ratio of PB to E3LB-L2 intermediate is about 1:0.5, about 1:0.75, about 1:2, or about 0.5:1.
1.3 general synthetic methods for coupling CIDE with L1 to prepare L1-CIDE
In certain embodiments, CIDE is contacted with L1 and a third base in a third suitable solvent to produce a solution. In certain embodiments, the contacting is performed at about (about 25 ℃) for about 2 hours. The solution may then optionally be purified to produce L1-CIDE.
In certain embodiments, the molar ratio of CIDE to L1 is about 1:4. In certain embodiments, the molar ratio of CIDE to L1 is about 1:1, 1:2, 1:3, 1:5, 1:6, 1:7, or about 1:8.
1.4 general Synthesis methods for coupling L1-CIDE with antibodies
In certain embodiments, the L1-CIDE is contacted with a thiol and a fourth suitable solvent to form a fourth solution. The solution is then contacted with an antibody to prepare a conjugate. In certain embodiments, the
In certain embodiments, the thiol is maleimide or 4-nitropyridine disulfide. In certain embodiments, a suitable solvent is selected from the group consisting of dimethylformamide, dimethylacetamide, and propylene glycol.
In certain embodiments, the molar ratio of L1-CIDE to thiol-reactive groups is from about 3:1 to about 20:1.
In certain embodiments, a solution comprising L1-CIDE, a thiol reactive group, and a suitable solvent is contacted with the antibody for about 1 to about 24 hours. In certain embodiments, a solution comprising L1-CIDE, a thiol-reactive group, and a suitable solvent is contacted with the antibody at about room temperature (about 25 ℃) to about 37 ℃.
In certain embodiments of the above general method, the suitable solvent is a polar aprotic solvent selected from the group consisting of dimethylformamide, tetrahydrofuran, ethyl acetate, acetone, acetonitrile, dimethyl sulfoxide, and propylene carbonate.
In certain embodiments of the general method described above, the base is selected from the group consisting of N, N-Diisopropylethylamine (DIEA), triethylamine, and 2,2,2,6,6-tetramethylpiperidine. In certain embodiments, the coupling agent is selected from the group consisting of: 1- [ bis (dimethylamino) methylene ] -1H-1,2, 3-triazolo [4,5-b ] pyridinium 3-oxohexafluorophosphate (HATU), (benzotriazol-1-yloxy) tris (dimethylamino) phosphonium hexafluorophosphate (BOP), (7-azabenzotriazol-1-yloxy) tripyrrolidine phosphorus hexafluorophosphate (PyAOP), O- (benzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HBTU), O- (benzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium tetrafluoroborate (TBTU), O- (6-chlorobenzotriazol-1-yl) -N, N, N '-tetramethyluronium Hexafluorophosphate (HCTU), O- (N-succinimidyl) -1, 3-tetramethyluronium tetrafluoroborate (TSTU), O- (5-norbornene-2, 3-dicarboxyimino) -N, N', N '-tetramethyluronium tetrafluoroborate (CDTU), O- (6-chlorobenzotriazol-1-yl) -N, N, N', N '-tetramethyluronium tetrafluoroborate (TBTU), O- (6-chlorobenzotriazol-1-yl) -N, N, N' -tetramethyluronium tetrafluoroborate (TBTU).
In a preferred embodiment, the solvent is dimethylformamide, the base is N, N-diisopropylethylamine, and the coupling agent is HATU.
In certain embodiments of the general method described above, the contacting is for about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 20 hours, 40 hours, 60 hours, or 72 hours.
In some embodiments of the above general method, the contacting is performed at about 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, 45 ℃, 46 ℃, 47 ℃, 48 ℃, 49 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃, or 100 ℃.
The following examples are provided by way of illustration only and not by way of limitation.
Examples
Unless otherwise indicated, all compounds are mixtures of olefin isomers (about 1:1). Listed in brackets 13 The C resonance represents the olefin isomer of the major isomer of a particular compound and/or the rotamer of the N-Me amide bond.
Synthesis example 1
Synthesis of L1-CIDE-BRM1-1
L1-CIDE-BRM1-1 is synthesized by the following scheme (scheme 1):
Figure BDA0004113777320001171
3,3' -disulfanediylbis (butan-2-ol) is synthesized in two steps:
Figure BDA0004113777320001172
to a solution of 3-mercaptobutan-2-ol (2.0 g,19 mmol) in anhydrous dichloromethane (40 mL) at 23℃was added MnO 2 (2.46 g,28 mmol). The reaction mixture was stirred at 23 ℃ for 1h and then filtered. The filtrate was concentrated in vacuo to give 3,3' -dithioalkanediylbis (butan-2-ol) (1.97 g, 99%) as a colourless oil. 1 H NMR(400MHz,CDCl 3 ) Delta 4.13-4.08 and 3.83-3.78 (m, 2H), 2.94-2.91 and 2.82-2.78 (m, 2H), 2.38-2.26 and 2.14-2.02 (m, 2H), 1.35-1.22 (m, 12H).
S- (3-hydroxybutyrin-2-yl) methylthiosulfonate, the following compound 3 (i.e., compound 2 in scheme 1 above) was synthesized as follows:
Figure BDA0004113777320001181
to a solution of 3,3' -disulfanediylbis (butan-2-ol) (1.97 g,9.36 mmol) in dry dichloromethane (50 mL) at 23℃was added sodium methanesulfonate (1.91 g,18.7 mmol) and iodine (2.38 g,9.36 mmol). The reaction mixture was stirred at 23℃for 1 day in the absence of light and then filtered. The filtrate was concentrated and the residue was purified by silica gel chromatography (eluting with 0 to 5% MeOH in DCM) to give S- (3-hydroxybut-2-yl) methylthiosulfonate (1.20 g, 70%) as a pale yellow oil. 1 H NMR(400MHz,CDCl 3 ) Delta 4.13-4.11 and 3.96-3.93 (m, 1H), 3.77-3.73 and 3.51-3.47 (m, 1H), 3.42 and 3.39 (s, 3H), 2.04 and 1.96 (brs, 1H), 1.53 and 1.42 (d, j=7.2 hz, 3H), 1.33 and 1.23 (d, j=6.0 hz, 3H).
Synthesis of S- (3- ((chlorocarbonyl) oxy) butan-2-yl) methylthiosulfonate.
Figure BDA0004113777320001182
To a solution of S- (3-hydroxybut-2-yl) methylthiosulfonate (300 mg,1.63 mmol) and pyridine (516 mg,6.51 mmol) in dichloromethane (2 mL) at 23℃was added a solution of triphosgene (242 mg,0.81 mmol) in dichloromethane (2 mL). The reaction was stirred at 23℃for 30 minutes. The reaction mixture was concentrated to dryness to give the title compound (380 mg, 95%) as a yellow oil, which was used directly in the next step.
S- (3- (((((3R, 5S) -1- ((R) -2- (3- (2, 2-diethoxyethoxy) isoxazol-5-yl) -3-methylbutanoyl) -5- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) carbonyl) oxy) butan-2-yl) methylthiosulfonate
To a solution containing (2S, 4R) -1- ((R) -2- (3- (2, 2-diethoxyethoxy) isoxazol-5-yl) -3-methylbutanoyl) -4-hydroxy-N- ((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) pyrrolidine-2-carboxamide [ compound 1 in the above example 1 scheme at 23 ℃; for the preparation process, see U.S. Pat. No. 5,2020/0038378, pages 293 to 294 (top of page number). ](300 mg,0.49 mmol) and
Figure BDA0004113777320001183
anhydrous dichloromethane (5 mL) of MS (100 mg)To the mixture was slowly added triethylamine (198 mg,1.95 mmol) and a solution of S- (3- ((chlorocarbonyl) oxy) butan-2-yl) methylthiosulfonate (362 mg,1.46 mmol) in anhydrous dichloromethane (2 mL) at 23 ℃. The mixture was stirred at 23 ℃ for 16 hours and then concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (0-70% ethyl acetate in petroleum ether) to give the title compound (120 mg, 30%) as a white solid. LCMS (ESI) m/z:825.3[ M+H ]] + 。
Synthesis of S- (3- (((((3R, 5S) -1- ((R) -3-methyl-2- (3- (2-oxoethoxy) isoxazol-5-yl) butanoyl) -5- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) carbonyl) oxy) butan-2-yl) methylthiosulfonate
Figure BDA0004113777320001191
To a solution of S- (3- (((((3R, 5S) -1- ((R) -2- (3- (2, 2-diethoxyethoxy) isoxazol-5-yl) -3-methylbutanoyl) -5- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) carbonyl) oxy) butan-2-yl) methylthiosulfonate (120 mg,0.15 mmol) was added a solution of formic acid (2 mL,2 mmol) in water (1 mL) at 23 ℃. The mixture was stirred at 50 ℃ for 1 hour, then concentrated to give the title compound (105 mg, 96%) as a yellow oil. LCMS (ESI) m/z:751.2[ M+H ] ] + 。
Synthesis of S- (3- (((((3R, 5S) -1- ((2R) -2- (3- (2- ((3R) -4- (2- ((4- (3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazin-1-yl) ethoxy) isoxazol-5-yl) -3-methylbutanoyl) -5- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) butan-2-yl) methylthiosulfonate
Figure BDA0004113777320001192
To a composition comprising S- (3- (((((3R, 5S) -1- ((R) -3-methyl-2- (3- (2-oxoethoxy) isoxazole)5-yl) butyryl) -5- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) butan-2-yl) methylthiosulfonate (105 mg,0.14 mmol) and 2- (6-amino-5- (8- (2- (2- ((R) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]Octan-3-yl) pyridazin-3-yl) phenol [ compound 3 of the above example 1 scheme. This is compound 4 in the scheme of example 4 below.]NaBH (OAc) was added to a 23℃solution of (73 mg,0.14 mmol) and HOAc (0.2-0.3 mL) in methylene chloride (2 mL) and methanol (2 mL) 3 (593 mg,2.80 mmol). The reaction mixture was stirred at 23 ℃ for 3 hours and then concentrated. The residue was purified by preparative TLC (8% methanol in dichloromethane) to give the title compound (48 mg, 27%) as a white solid. 1 H NMR(400MHz,DMSO-d 6 )：δ14.21-14.08(m,1H),9.01-8.97(m,1H),8.59-8.46(m,1H),7.92(d,J＝4.8Hz,1H),7.79(d,J＝6.0Hz,1H),7.55-7.33(m,5H),7.28-7.19(m,1H),6.96-6.79(m,2H),6.60-6.48(m,1H),6.18-6.08(m,2H),6.05-5.91(m,2H),5.24-5.11(m,1H),4.99-4.86(m,2H),4.57-4.44(m,2H),4.43-4.35(m,1H),4.31-4.17(m,4H),3.94-3.79(m,2H),3.77-3.67(m,2H),3.61-3.51(m,2H),3.29-3.23(m,4H),3.21-3.14(m,3H),3.04-2.93(m,3H),2.87-2.78(m,1H),2.64-2.58(m,3H),2.48-2.41(m,3H),2.38-2.31(m,2H),2.20-2.16(m,2H),2.07-1.82(m,2H),1.49-1.21(m,10H),1.06-0.90(m,6H),0.88-0.76(m,3H)；LCMS(ESI)m/z：1251.0[M+H] + 。
Synthesis example 2
Synthesis of L1-CIDE-BRM1-2
L1-CIDE-BRM1-2 is synthesized by the following scheme (scheme 2):
Figure BDA0004113777320001211
synthesis of tert-butyl (2S, 4R) -2- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) -4- (((4-nitrophenoxy) carbonyl) oxy) pyrrolidine-1-carboxylate
Figure BDA0004113777320001212
To a mixture of 4-nitrophenyl chloroformate (1.68 g,8.34 mmol) and (2S, 4R) -4-hydroxy-2- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidine-1-carboxylic acid tert-butyl ester (see compound 1 in example scheme 2 above; see J.Med. Chem.2019,62,941 or J.Med. Chem.2014,57,8657) (3.0 g,6.95 mmol) in dry dichloromethane (80 mL) at 23℃was added 2, 6-dimethylpyridine (1.12 g,10.4 mmol). The reaction mixture was stirred at 23 ℃ for 18 hours and then concentrated to give the title compound (4.0 g, 99%) as a yellow solid. The material was used directly in the next step. LCMS (ESI) m/z:597.2[ M+H ]] + 。
Compound 4, scheme 2: synthesis of allyl 1- (((2S) -1- ((4- (1-hydroxy-2- (4-methylpiperazin-1-yl) -2-oxoethyl) phenyl) amino) -1-oxo-5-ureidopent-2-yl) carbamoyl) cyclobutanecarboxylate by scheme 2a
Figure BDA0004113777320001221
i. General procedure for preparation of Compound 2 of scheme 2a
Figure BDA0004113777320001231
Four separate reactions were performed in parallel. SeO was added to a pyridine (3.00L) solution containing Compound 1 (200 g,1.21 mol) at 23 ℃ 2 (336 g,3.03 mol). The mixture was then heated in an oil bath at 95 ℃ for 1 hour. The four reactions were combined for work-up. The combined reactions were filtered at 45-50 ℃ and the filtrate was then cooled to 23 ℃ and held at that temperature for 1.5 hours. The mixture was filtered and the filter cake was dried in vacuo to give compound 2. The filtrate was concentrated to 5.00L under reduced pressure and stirred at 23℃for 12 hours. The mixture was filtered and the filter cake was dried in vacuo to give additional compound 2 as a yellow solid (two batches combined = 830g, 88% yield). 1 H NMR(400MHz,DMSO-d 6 ):δ8.63-8.64(m,2H),8.37-8.40(m,2H),8.17-8.19(m,2H),7.90-7.94(m,1H),7.48-7.52(m,2H)。
General procedure for preparation of Compound 3 of scheme 2a
Figure BDA0004113777320001232
The reactions were performed in duplicate. To a solution of compound 2 (140 g, 178 mmol) in DMF (700 mL) at 23deg.C was added compound 2A (54 g, 178 mmol), HATU (225 g, 225 mmol) and DIPEA (278 g,2.15 mol). The mixture was stirred at 23℃for 1 hour. The two reactions were then combined for work-up. The combined reaction mixtures were diluted with DCM (3.00L) and washed with brine (1.00L x 3). The organic layer was taken up with Na 2 SO 4 Dried, filtered and concentrated under reduced pressure. Purification of the residue by column chromatography (SiO 2 Petroleum ether/ethyl acetate=50/1 to 0/1) to give compound 3 (200 g, 67% yield) as a yellow solid.
General procedure for preparation of Compound 4 of scheme 2a
Figure BDA0004113777320001233
NaBH was added to a solution of Compound 3 (184 g,531 mmol) in MeOH (1.30L) at 0deg.C 4 (16.1 g,425 mmol). The mixture was warmed to 23 ℃ and stirred at that temperature for 1 hour. The reaction mixture was concentrated under reduced pressure and the residue was diluted with water, adjusted to ph=7 with HCl (1M) and extracted with EtOAc (1.00 l x 3). The combined organic layers were taken up in Na 2 SO 4 Dried, filtered, and concentrated under reduced pressure. Purification of the residue by column chromatography (SiO 2 Dichloromethane/methanol=100/1 to 10/1). The crude product was triturated with EtOH (500 mL) at 23 ℃ for 10 min to give compound 4 as a yellow solid (161 g, 54% yield). 1 H NMR：(400MHz,DMSO-d 6 ):δ8.24(d,J＝8.4Hz,2H),7.64(d,J＝8.4Hz,2H),6.14(d,J＝6.4Hz,1H),5.60(d,J＝6.0Hz,1H),3.57-3.47(m,2H),3.43(s,2H),2.23(s,2H),2.12(s,5H)。
General procedure for preparation of Compound 5 of scheme 2a
Figure BDA0004113777320001241
Four reactions were performed in parallel. At N 2 Pd/C (8.50 g, 10%) was added to a solution of compound 4 (40 g,143 mmol) in EtOH (600 mL) under an atmosphere. Degassing the suspension and using H 2 Purging 3 times. The mixture is put in H 2 (15 psi) at 23℃for 12 hours. The four reactions are then combined for work-up. The combined reaction mixtures were filtered and the filter cake was washed with MeOH (1.00L). The combined filtrate and washings were concentrated under reduced pressure to give compound 5 (140 g, yield 98%) as a yellow solid. 1 H NMR：(400MHz,CD 3 OD):δ7.11(d,J＝8.4Hz,2H),6.72(d,J＝8.4Hz,2H),5.29(s,1H),3.74(s,1H),3.62-3.49(m,1H),3.47-3.35(m,2H),2.48(s,1H),2.35-2.25(m,2H),2.22(s,3H),1.90(s,1H)。
General procedure for preparation of Compound 6 of scheme 2a
Figure BDA0004113777320001242
To a solution of Fmoc-L-citrulline (compound 5A) (95 g,239 mmol) and compound 5 (72 g,287 mmol) in MeOH (350 mL) and DCM (700 mL) at 0deg.C was added one portion of EEDQ (71 g,287 mmol). The mixture was warmed to 23℃and heated to N 2 Stirring was carried out at this temperature for 15 hours. The reaction mixture was concentrated under reduced pressure. The crude product was triturated with MTBE (1.00L) at 15 ℃ for 2 hours to give compound 6 (185 g, crude product) as an orange solid.
General procedure for preparation of Compound 7 of scheme 2a
Figure BDA0004113777320001251
Piperidine (52 g,604 mmol) was added to a solution of compound 6 (190 g,302 mmol) in DCM (1.40L) at 10deg.C. The mixture was stirred at 10 ℃ for 18 hours and then concentrated under reduced pressure. By column chromatographyPurification of the residue (SiO) 2 Dichloromethane/methanol=100/1 to 3/1) to give compound 7 (85 g, yield 68%) as a yellow oil. 1 H NMR(400MHz,CD 3 OD):δ7.65(d,J＝8.4Hz,2H),7.37(d,J＝8.4Hz,2H),5.43(s,1H),3.68(s,1H),3.62(d,J＝4.0Hz,1H),3.53-3.44(m,2H),3.24-3.06(m,2H),2.81(d,J＝5.2Hz,2H),2.45(s,1H),2.36-2.25(m,2H),2.22(s,3H),1.97(s,1H),1.86-1.75(m,1H),1.68-1.54(m,6H)。
General procedure for preparation of Compound 8 of scheme 2a
Figure BDA0004113777320001252
To 10℃DME (470 mL) and H containing Compound 7 (76 g,187 mmol) 2 To a solution of O (290 mL) was added Compound 7A (63 g,234 mmol) and NaHCO 3 (20 g,234 mmol). The mixture was stirred at 10 ℃ for 12h and then concentrated under reduced pressure. Purification of the residue by column chromatography (SiO 2 Dichloromethane/methanol=10/1 to 3/1) to give compound 8 (92 g, yield 70%) as a yellow solid.
General procedure for preparation of Compound 9 of scheme 2a
Figure BDA0004113777320001261
To a stirred solution of compound 8 (63 g,112 mmol) in THF (190 mL) and MeOH (95 mL) at 0deg.C was added LiOH H 2 O (9.43 g,225 mmol) H 2 O (190 mL) solution. The reaction mixture was warmed to 15 ℃ and stirred at that temperature for 12 hours. The reaction mixture was then concentrated under reduced pressure. The residue was purified by reverse phase HPLC (0.1% TFA conditions) to give compound 9 (50 g, 81% yield) as a white solid. 1 H NMR(400MHz,CD 3 OD):δ7.68(d,J＝7.6Hz,2H),7.37(d,J＝8.4Hz,2H),5.48(s,1H),4.50-4.53(m,1H),3.78(s,2H),3.69-3.56(m,1H),3.27-3.10(m,5H),2.79(s,3H),2.71-2.62(m,2H),2.60-2.50(m,2H),2.17-2.07(m,1H),2.06-2.03(m,4H),2.03-1.97(m,1H),1.97-1.86(m,1H),1.74-1.78(m,1H),1.69-1.53(m,2H)。
General procedure for preparation of Compound 4 of scheme 2 (220)
Figure BDA0004113777320001262
To a solution of Compound 9 (70 g,131 mmol) in DMF (350 mL) at 15deg.C was added KF (23 g, 390 mmol) and Bu 4 NHSO 4 (12.5 g,36.8 mmol). 3-Bromoprop-1-ene (390 g,3.29 mol) was then added dropwise at 15℃and the mixture was stirred at this temperature for a further 6 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: phenomenex luna c, 250 mm. Times.100 mm. Times.10 um; mobile phase: [ water (0.1% TFA) -ACN]The method comprises the steps of carrying out a first treatment on the surface of the B%:0% -20%,30 min) to give 220 as a white solid (26 g, 34% yield). 1 H NMR(400MHz,CD 3 OD):δ7.69(d,J＝8.4Hz,2H),7.39(d,J＝8.4Hz,2H),6.02(s,1H),5.75(d,J＝10.0Hz,2H),5.49(s,1H),4.50-4.54(m,1H),4.34-4.09(m,1H),4.03(s,2H),3.96-3.51(m,3H),3.50-3.33(m,3H),3.26-3.06(m,6H),2.75-2.49(m,4H),2.22-2.08(m,1H),2.06-1.87(m,2H),1.81-1.72(m,1H),1.70-1.52(m,2H)。LCMS：(M+H + ＝573.3)
Compound 6, scheme 2: synthesis of 1- (((2S) -1- ((4- (1- (((((3R, 5S) -1- (tert-butoxycarbonyl) -5- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) carbonyl) oxy) -2- (4-methylpiperazin-1-yl) -2-oxoethyl) phenyl) amino) -1-oxo-5-ureidopent-2-yl) carbamoyl) cyclobutanecarboxylic acid
Figure BDA0004113777320001271
To a catalyst containing (2S, 4R) -4- (((1- (4- ((S) -2- (1- ((allyloxy) carbonyl) cyclobutanecarboxamido) -5-ureidovaleramido) phenyl) -2- (4-methylpiperazin-1-yl) -2-oxoethoxy) carbonyl) oxy) -2- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidine-1-carboxylic acid tert-butyl ester (76 mg,0.07 mmol) and 1, 3-dimethylpyrimidine-2, 4,6 (1H, 3H, 5H) -trione (58 mg,0.37 mmol) at 23℃Pd (PPh) was added to a solution of methane (5 mL) and methanol (5 mL) 3 ) 4 (17 mg,0.01 mmol). The reaction mixture was stirred at 23 ℃ under nitrogen atmosphere for 10 hours and then concentrated. The residue was purified by preparative HPLC (chromatographic conditions as follows: column Phenomenex Gemini-NX 80X 30mM 3um, mobile phase (25-45%) water (10 mM NH) 4 HCO 3 ) ACN) to give the title compound (50 mg, 69%) as a yellow solid. LCMS (ESI) m/z:990.6[ M+H ]] + 。
Synthesis of tert-butyl (2S, 4R) -4- (((1- (4- ((S) -2- (1- ((5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) carbamoyl) cyclobutanecarboxamido) -5-ureidovaleramido) phenyl) -2- (4-methylpiperazin-1-yl) -2-oxoethoxy) carbonyl) oxy) -2- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidine-1-carboxylate
Figure BDA0004113777320001281
N, N-diisopropylethylamine (0.03 mL,0.21 mmol) and HATU (32 mg,0.08 mmol) were added to a mixture of 1- (((2S) -1- ((4- (1- (((((3R, 5S) -1- (tert-butoxycarbonyl) -5- (((S) -1- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) carbonyl) oxy) -2- (4-methylpiperazin-1-yl) -2-oxoethyl) phenyl) amino) -1-oxo-5-ureidopent-2-yl) carbamoyl) cyclobutanecarboxylic acid (69 mg,0.07 mmol) and 1- (5-aminopentyl) -1H-pyrrole-2, 5-dione (16 mg,0.08 mmol) in DMF (8 mL) at 23 ℃. The reaction mixture was stirred at 23 ℃ for 16 hours and then concentrated. The residue was purified by preparative HPLC (Boston Green ODS 150 x 30mm x 5um, (25-45%) water (0.075% tfa) -ACN) to give the title compound (67 mg, 84%) as a white solid. LCMS (ESI) m/z:1155.6[ M+H ]] + 。
Compound 7, scheme 2: synthesis of 2, 2-trifluoroacetate ester of 1- (4- ((S) -2- (1- ((5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) carbamoyl) cyclobutanecarboxamido) -5-ureidovaleramido) phenyl) -2- (4-methylpiperazin-1-yl) -2-oxoethyl ((3R, 5S) -5- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-3-yl) carbonate
Figure BDA0004113777320001282
A solution of (2S, 4R) -4- (((1- (4- ((S) -2- (1- ((5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) carbamoyl) cyclobutanecarboxamido) -5-ureidovaleramido) phenyl) -2- (4-methylpiperazin-1-yl) -2-oxoethoxy) carbonyl) oxy) -2- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidine-1-carboxylic acid tert-butyl ester (67.4 mg,0.06 mmol) in HFIP (2 mL,0.06 mmol) of 5% TFA was stirred at 23℃for 1.5H. The reaction mixture was concentrated to give the title compound (62 mg, 99.9%) as a yellow oil. LCMS (ESI) m/z:1054.7[ M+H ] ] + 。
L1-CIDE-BRM1-2: synthesis of (3R, 5S) -1- ((2R) -2- (3- ((3R) -4- (2- ((4- (3- (3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) oxy) pyridin-2-yl) oxy) ethyl) -3-methylpiperazin-1-yl) ethoxy) isoxazol-5- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-3-yl (1- (4- ((S) -2- (1- ((5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) carbamoyl) cyclobutanecarboxamide) -5-ureidopentanamido) phenyl) -2- (4-methylpiperazin-1-yl) -2-oxoethyl) carbonate
Figure BDA0004113777320001291
To a catalyst containing 1- (4- ((S) -2- (1- ((5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) carbamoyl) cyclobutanecarboxamido) -5-ureidovaleramido) phenyl) -2- (4-methylpiperazin-1-yl) -2-oxoethyl ((3R, 5S) -5- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-3-yl) carbonate 2, 2-trifluoroacetate (62 mg,0.06 mmol) and (2R) -2- (3- (2- ((3R) -4- (2- ((4- (3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]Octan-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazin-1-yl) ethoxy) isoxazol-5-yl) -3-methylbutan To a mixture of acids (50 mg,0.07 mmol) in DMF (2.5 mL) was added N, N-diisopropylethylamine (0.03 mL,0.18 mmol) and HATU (27 mg,0.07 mmol). The reaction mixture was stirred at 23 ℃ for 16 hours and then concentrated. The residue was purified by preparative HPLC (chromatographic conditions as follows: column Phenomenex Gemini-NX 80X 30mM 3um; mobile phase (26-46%) water (10 mM NH) 4 HCO 3 ) ACN) to give the title compound (44 mg, 42%) as a white solid. 1 H NMR(400MHz,CD 3 OD):δ8.88-8.83(m,1H),7.80-7.75(m,1H),7.75-7.65(m,3H),7.49-7.32(m,7H),7.25-7.19(m,1H),6.93-6.84(m,2H),6.76(s,1H),6.75-6.71(m,1H),6.57-6.54(m,1H),6.29-6.19(m,2H),5.25-5.21(m,1H),4.98-4.93(m,2H),4.64-4.62(m,2H),4.51(s,3H),4.41-4.27(m,3H),4.23-4.08(m,1H),4.01-3.89(m,1H),3.79-3.54(m,4H),3.48-3.40(m,2H),3.27-3.16(m,4H),3.15-3.03(m,4H),2.97-2.85(m,2H),2.83-2.69(m,4H),2.61-2.51(m,3H),2.50-2.33(m,8H),2.29-2.17(m,6H),2.14-2.11(m,3H),1.93(s,4H),1.75(s,1H),1.62-1.45(m,9H),1.35-1.23(m,4H),1.16-1.10(m,3H),1.09-0.92(m,3H),0.92-0.80(m,3H)；LCMS(ESI)m/z：1764.8[M+H] + 。
Synthesis example 3
Synthesis of L1-CIDE-BRM1-3
L1-CIDE-BRM1-3 is synthesized by the following scheme (scheme 3):
Figure BDA0004113777320001301
synthesis of S- (3- ((chlorocarbonyl) oxy) butan-2-yl) methylthiosulfonate
Figure BDA0004113777320001302
To a solution of S- (3-hydroxybut-2-yl) methylthiosulfonate (300 mg,1.63 mmol) in dichloromethane (2 mL) and pyridine (516 mg,6.51 mmol) at 23℃was added a solution of triphosgene (242 mg,0.81 mmol) in dichloromethane (2 mL). The reaction was stirred at 23 ℃ for 30min and then concentrated to dryness to give the title compound (380 mg, 95%) as a yellow oil. The material was used directly in the next step.
Synthesis of S- (3- (((((3R, 5S) -1- ((R) -2- (3- (4- (dimethoxymethyl) piperidin-1-yl) isoxazol-5-yl) -3-methylbutanoyl) -5- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) carbonyl) oxy) butan-2-yl) methylthiosulfonate
Figure BDA0004113777320001311
To 23 ℃ (2S, 4R) -1- ((R) -2- (3- (4-methoxymethyl) piperidin-1-yl) isoxazol-5-yl) -3-methylbutanoyl-4-hydroxy-N- ((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) pyrrolidine-2-carboxamide (250 mg,0.39 mmol) (see compound 1 in scheme 3 above; see U.S. Pat. No. 5,2020/0038378 pages 451-452 for a description of the preparation methods thereof, which is incorporated herein by reference in its entirety) and
Figure BDA0004113777320001313
to a mixture of MS (50 mg) in dichloromethane (2 mL) was added pyridine (0.09 mL,1.17 mmol) and a solution of S- (3- ((chlorocarbonyl) oxy) butan-2-yl) methylthiosulfonate (220 mg,0.89 mmol) in dichloromethane (1 mL). The reaction was stirred at 23 ℃ for 30 minutes and then concentrated. The residue was purified by flash chromatography on a silica gel column (ethyl acetate containing 0-60% methylene chloride) to give the title compound 3 (130 mg, 39%) as a white solid. LCMS (ESI) m/z:850.3[ M+H ]] + 。
Synthesis of S- (3- (((((3R, 5S) -1- ((R) -2- (3- (4-formylpiperidin-1-yl) isoxazol-5-yl) -3-methylbutanoyl) -5- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) carbonyl) oxy) butan-2-yl) methylthiosulfonate
Figure BDA0004113777320001312
Will contain S- (3- (((((3R, 5S) -1- ((R) -2- (3- (4- (dimethoxymethyl) piperidin-1-yl) isoxazol-5-yl) -3-methylbutanoyl) -5- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidine-3) -yl) oxy) carbonyl) oxy) butan-2-yl) methylthiosulfonate (130 mg,0.15 mmol) in water (1 mL) and formic acid (3 mL) were stirred at 50 ℃ for 2 hours. The reaction mixture was then concentrated to give the title compound (120 mg, 98%) as a yellow oil. LCMS (ESI) m/z:804.3[ M+H ]] + 。
L1-CIDE-BRM1-3: synthesis of S- (3- (((((3R, 5S) -1- ((2R) -2- (3- ((4- (trans-3- ((4- (3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) cyclobutoxy) piperidin-1-yl) methyl) piperidin-1-yl) isoxazol-5-yl) -3-methylbutyl) -5- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) carbonyl) butan-2-yl) methylthiosulfonate
Figure BDA0004113777320001321
To a solution of S- (3- (((((3R, 5S) -1- ((R) -2- (3- (4-formylpiperidin-1-yl) isoxazol-5-yl) -3-methylbutanoyl) -5- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) carbonyl) butan-2-yl) methylthiosulfonate (120 mg,0.15 mmol) in dichloromethane (1 mL) and methanol (1 mL) at 23 ℃ was added 2- (6-amino-5- (8- (2- (trans-3- (piperidin-4-yloxy) cyclobutoxy) piperidin-4-yl) -3, 8-diazabicyclo [ 3.2.1) ]Octan-3-yl) pyridazin-3-yl) phenol hydrochloride (see compound 5 in scheme 3 above; preparation is described in U.S. Pat. No. 5,2020,0038378, pages 306-307 (top of page number.) (82 mg,0.15 mmol), HOAc (0.2 ml) and sodium triacetoxyborohydride (317 mg,1.49 mmol). The reaction mixture was stirred at 23 ℃ for 3 hours and then concentrated. The crude residue was purified by preparative TLC (methanol: dichloromethane=1:10) to give the title compound (31 mg, 14%) as a white solid. 1 H NMR(400MHz,DMSO-d 6 ):δ14.13(s,1H),8.98(s,1H),8.49(d,J＝7.2Hz,1H),7.91(d,J＝7.2Hz,1H),7.76(d,J＝6.4Hz,1H),7.52-7.41(m,3H),7.40-7.33(m,2H),7.24-7.21(m,1H),6.90-6.80(m,2H),6.54-6.51(m,1H),6.14-6.12(m,2H),6.00-5.91(m,2H),5.18-5.15(m,2H),4.95-4.90(m,2H),4.50-4.46(m,2H),4.39-4.35(m,1H),4.32-4.22(m,1H),3.94-3.66(m,3H),3.64-3.55(m,4H),3.54-3.52(m,2H),3.28-3.21(m,4H),3.02-3.01(m,2H),2.77-2.69(m,2H),2.45(s,3H),2.30-2.22(m,4H),2.19-2.15(m,2H),2.10-2.05(m,2H),2.04-1.89(m,5H),1.81-1.57(m,5H),1.47-1.34(m,8H),1.33-1.28(m,2H),1.27-1.20(m,2H),1.16-1.02(m,2H),0.95-0.91(m,3H),0.85-0.75(m,3H)；LCMS(ESI)m/z：1331.9[M+H] + 。
Synthesis example 4
Synthesis of L1-CIDE-BRM1-4
L1-CIDE-BRM1-4 is synthesized by the following scheme (scheme 4):
Figure BDA0004113777320001331
/>
Figure BDA0004113777320001341
compound 1, scheme 4:
step 1: 182.
Figure BDA0004113777320001342
2-fluoro-4-iodopyridine (2) (52 g,230 mmol) was added to 3, 8-diazabicyclo [ 3.2.1-containing]A single neck 2L round bottom flask of a magnetically stirred mixture of tert-butyl octane-3-carboxylate (1) (37 g,175 mmol), sodium tert-butoxide (26 g,265 mmol), potassium fluoride (17 g,284 mmol) and xantphos (4.8 g,8.2 mmol) in 1, 4-dioxane (750 mL). The mixture was purged with nitrogen for 15 minutes, then tris (dibenzylideneacetone) dipalladium (0) (3.7 g,4.0 mmol) was added. The reaction flask was equipped with a condenser with nitrogen inlet and placed in an oil bath preheated to 110 ℃. After stirring at 110 ℃ for 1.25 hours under nitrogen atmosphere, the resulting brown/red suspension was cooled to 23 ℃ and filtered through celite. With Et 2 The cake was washed with O and the combined filtrate and washings were concentrated under reduced pressure to a red oil (127 g). The crude material was purified by flash chromatography (using 0-40% etoac/DCM) to give 182 as a yellow foamy solid (56 g, about 100%).
Step 2: preparation of 187.
Figure BDA0004113777320001343
A solution of 4M HCl in 1, 4-dioxane (220 mL, 88mmol) was added over 50 minutes to a magnetically stirred solution of 182 (56 g, about 175 mmol) MeCN (650 mL) in a single neck 2L round bottom flask at 23 ℃. The mixture was stirred at 23 ℃ for 1 hour, during which time the mixture turned into a yellow/orange suspension. The reaction mixture was concentrated to a yellow solid and the material was taken up in Et at 23 ℃ 2 O (1000 mL) was milled for 1 hour. The mixture was filtered, and the collected material was dried under reduced pressure to give tri-HCl salt 187 (61 g) as a yellow powder. The salt was suspended in DCM (1000 mL) and saturated NaHCO 3 The aqueous solution (500 mL) was slowly neutralized. The layers were separated and the aqueous phase was further extracted with DCM (2X 500 mL). The combined organic phases were washed with saturated NaCl (250 mL), dried over anhydrous Na 2 SO 4 Dried, filtered and concentrated to give the free base as a yellow solid 187 (32 g, 86%). Note that: ensure NaHCO 3 The solution was saturated enough to prevent loss of product in the aqueous phase.
Step 3: 208.
Figure BDA0004113777320001351
1, 8-diazabicyclo [5.4.0] undec-7-ene (5) (3.0 mL,20 mmol) was added to a magnetically stirred solution of 187 (30 g,145 mmol) 3-amino-4-bromo-6-chloropyridazine (44 g,209 mmol) and N, N-diisopropylethylamine (80 mL,460 mmol) in anhydrous DMF (300 mL) in a 1L Erlenmeyer flask at 23 ℃. The solution was uniformly distributed into two 450mL sealable round bottom flasks and then magnetically stirred in an oil bath set at 100 ℃ for 23 hours. The clear red reaction mixtures were combined and concentrated under high vacuum to a brown residue (107 g). The residue was purified twice by flash chromatography (using 0-5% meoh/DCM) to give a complex of 208 as a yellow solid with one equivalent of N, N-diisopropylethylamine (20 g, 30%). The complex contains about 72 wt% 208 (about 15g 208).
Step 4: 04-1.
Figure BDA0004113777320001352
2-hydroxyphenyl boric acid (9.0 g,65 mmol) was added to a single neck 2L round bottom flask containing a magnetically stirred mixture of 208 amine complex (17 g,37 mmol) and potassium carbonate (16 g,113 mmol) in a mixture of 1, 4-dioxane (600 mL) and deionized water (120 mL) at 23 ℃. The mixture was purged with nitrogen for 30 minutes, then RuPhos-Pd-G3 (1.8G, 2.1 mmol) was added. The flask was equipped with a condenser with nitrogen inlet and placed in an oil bath preheated to 100 ℃. After stirring at 100 ℃ under nitrogen for 23 hours, the resulting dark red solution was cooled to 23 ℃ and concentrated under reduced pressure to a brown solid (40 g). The crude material 1 H NMR analysis showed 04-1 as the main product. The above material was combined with 6.8g crude 04-1 of similar purity from the earlier batch. The combined batches were purified by flash chromatography (using 0-100% EtOAc/DCM) followed by further chromatography (eluting with 0-10% MeOH/DCM) to give 04-1 as a yellow solid with 98% purity (measured by HPLC). Et for solid 2 O was triturated, collected by filtration, and dried under reduced pressure to give 04-1 as a yellow powder (8.0 g, 47% combined yield).
Compound 3, scheme 4: synthesis of tert-butyl (3R) -4- (2- ((4- (3- (3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazine-1-carboxylate
Figure BDA0004113777320001361
To a compound containing 2- (6-amino-5- (8- (2-fluoropyridin-4-yl) -3, 8-diazabicyclo [3.2.1] at 23 DEG C]Octan-3-yl) pyridazin-3-yl phenol (1.5 g,3.82 mmol) (see Compound 1 in scheme 4 above) and sodium hydride (60% in mineral oil solution)In (a) and (b); to a solution of 0.46g,11.47 mmol) in THF (20 mL) was added dropwise (R) -4- (2-hydroxyethyl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester (see compound 2 in scheme 4 above; see also WO2011/28685, page 88, column 89, the entire disclosure of which is incorporated herein by reference (1.87 g,7.64 mmol). The mixture was then stirred at 60℃for 12 hours. After cooling to 23 ℃, the reaction was diluted with water (100 mL) and the resulting mixture was extracted with ethyl acetate (150 ml×3). The combined organic layers were washed with brine (100 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and the residue purified by silica gel column flash chromatography (0-5% methanol in DCM) to give the title compound (1.2 g, 64%) as a grey solid. LCMS (ESI) m/z:617.6[ M+H ] ] + 。
Compound 4, scheme 4: synthesis of 2- (6-amino-5- (8- (2- (2- ((R) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenol
Figure BDA0004113777320001371
To a compound comprising (3R) -4- (2- ((4- (3- (3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]To a solution of tert-butyl octan-8-yl) pyridin-2-yloxy) -ethyl-3-methylpiperazine-1-carboxylate (1.2 g,1.95 mmol) in ethyl acetate (10 mL) at 23℃was added 4M HCl/EtOAc (20 mL,1.95 mmol). The mixture was stirred at 23 ℃ for 16 hours and then concentrated. The residue was purified by flash chromatography on a silica gel column (0-10% MeOH (1% NH) 3 ·H 2 DCM of O)) to give the title compound as a yellow solid (900 mg, 90%).
Synthesis of methyl 2- (3- (2- ((3R) -4- (2- ((4- (3- (3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazin-1-yl) ethoxy) isoxazol-5-yl) -3-methylbutanoate
Figure BDA0004113777320001372
At 23 DEG CTo a solution containing NaBH (OAc) 3 (746 mg,3.48 mmol), 2- (6-amino-5- (8- (2- (2- ((R) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]Octan-3-yl) pyridazin-3-yl phenol (900 mg,1.74 mmol) and methyl 3-methyl-2- (3- (2-oxoethoxy) isoxazol-5-yl) butyrate (see compound 5 in scheme 4 above; see U.S. Pat. No. 2020/0038378, page 428, incorporated herein by reference in its entirety, for a solution of (463 mg,1.92 mmol) in methanol (10 mL) and dichloromethane (10 mL) to which sodium acetate (710 mg,8.71 mmol) is added. The reaction mixture was stirred at 23 ℃ for 16 hours and then concentrated. The residue was purified by flash chromatography on a silica gel column (DCM containing 0-10% MeOH) to give the title compound (1.0 g, 77%) as a yellow solid. LCMS (ESI) m/z:742.6[ M+H ] ] + 。
Compound 6, scheme 4: synthesis of 2- (3- (2- ((3R) -4- (2- ((4- (3- (3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazin-1-yl) ethoxy) isoxazol-5-yl) -3-methylbutanoic acid
Figure BDA0004113777320001381
To methyl-containing 2- (3- (2- ((3R) -4- (2- ((4- (3- (3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]To a solution of octan-8-yl-pyridin-2-yloxy) ethyl) -3-methylpiperazin-1-yl-ethoxy-isoxazol-5-yl) -3-methylbutanoate (1.0 g,1.35 mmol) in water (6 mL) and methanol (6 mL) at 23℃was added lithium hydroxide monohydrate (3 mg,6.74 mmol). The mixture was stirred at 23 ℃ for 16 hours, then concentrated to give the title compound (980 mg) as a yellow solid. LCMS (ESI) m/z:728.3[ M+H ]] + 。
Compound 7, scheme 4: synthesis of (2R) -2- (3- (2- ((3R) -4- (2- ((4- (3- (3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazin-1-yl) ethoxy) isoxazol-5-yl) -3-methylbutanoic acid
Figure BDA0004113777320001382
By chiral SFC (DAICEL CHIRALPAK AD (250 mm. Times.50 mm,10 um) 0.1% NH 3 H 2 O, IPA, 18%) isolation of 2- (3- (2- ((3R) -4- (2- ((4- (3- (3-amino-6- (2-hydroxyphenyl) isoxazol-4-yl) -3, 8-diazabicyclo [ 3.2.1) ]Octane-8-yl) isoxazol-2-yl) oxy) ethyl) -3-methylpiperazin-1-yl) ethoxy-isoxazol-5-yl) -3-methylbutanoic acid (900 mg,1.24 mmol) to give the first peak (2S) -2- (3- (2- ((3R) -4- (2- ((4- (3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]Octan-8-yl) pyridin-2-yloxy) ethyl) -3-methylpiperazin-1-yl) ethoxy isoxazol-5-yl) -3-methylbutanoic acid (400 mg, 44%) and a second peak (2R) -2- (3- (2- ((3R) -4- (2- ((4- (3- (3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]Octane-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazin-1-yl) ethoxy) isoxazol-5-yl) -3-methylbutanoic acid (450 mg, 50%) both as white solids.
Synthesis of compound 15, scheme 4:
Figure BDA0004113777320001391
preparation of Compound 8 (hereinafter referred to as Compound Y) of scheme 4. Synthesis of allyl (((S) -1- ((2S, 4R) -4-hydroxy-2- ((4- (4-methylthiazol-5-yl) benzyl) carbamoyl) pyrrolidin-1-yl) -3, 3-dimethyl-1-oxobutan-2-yl) carbamate.
Allyl chloroformate (481mg, 4.02 mmol) was added to 0 ℃ containing (2S, 4 r) -4-hydroxy-N- ((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) pyrrolidine-2-carboxamide (see e.g. compound 1 above; see e.g. j. Med. Chem.2019,62,941) (1.65 g,3.83 mmol) and NaHCO for a description of the preparation process 3 (1.61 g,19.2 mmol) 1:1THF:H 2 In a mixture of O (34 mL). The resulting mixture was warmed to 25 ℃ and stirred at that temperature for 12 hours. After dilution with water (50 mL), the reaction mixture was extracted with EtOAc (3 x 50 mL) and the combined organic layers were washed with brine (50 mL), over Na 2 SO 4 Dried, filtered, and concentrated. By means of silica gel columnsThe residue was purified by flash chromatography (eluting with 0-3% MeOH in DCM) to give (2S, 4 r) -4-hydroxy-2- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidine-1-carboxylic acid allyl ester (see compound Y above) (1.60 g, 81%) as a grey solid. LCMS (10-80, ab,7.0 min): rt=2.57 min, m/z=537.1 [ m+na ]] + 。
Preparation of compound 9 of scheme 4. (2S, 4R) -4- ((hydroxyhydrophosphoryl) oxy) -2- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidine-1-carboxylic acid allyl ester
Figure BDA0004113777320001401
To a solution of allyl (2S, 4 r) -4-hydroxy-2- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidine-1-carboxylate (2.0 g,4.81 mmol) in THF (30 mL) at-78 ℃ was added PCl-containing 3 (1.67 mL,19.3 mmol) THF (5 mL) and Et-containing 3 N (4.03 mL,29 mmol) THF (3 mL). The reaction mixture was stirred at-78 ℃ for 20 minutes and then warmed to 23 ℃. The resulting mixture was stirred at 23℃for 12 hours, then with water (20 mL) and NaHCO 3 (5 mL) aqueous quenching. After stirring at 23 ℃ for 10 minutes, the mixture was acidified with 1N HCl to ph=3, then concentrated under reduced pressure. The residue was purified by flash chromatography on a silica gel column (DCM with 0-10% methanol) to give the title compound (1.5 g, 65%) as a colourless solid. LCMS (ESI) m/z:480.2[ M+H ]] + 。
Preparation of compound 10 of scheme 4. Synthesis of allyl (2S, 4R) -4- ((hydroxy (1H-imidazol-1-yl) phosphoryl) oxy) -2- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidine-1-carboxylate
Figure BDA0004113777320001403
Allyl (2S, 4R) -4- ((hydroxyhydrophosphoryl) oxy) -2- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidine-1-carboxylate (600 mg,1.25 mmol) and Et at 23 ℃ C 3 N (0.52 mL,3.75 mmol) CCl 4 To a solution of (8 mL) and acetonitrile (8 mL) at 23℃was added 1- (trimethylsilyl) -1H-imidazole (0.53 g,3.75 mmol). The reaction mixture was stirred at 23 ℃ for 40 minutes and then concentrated. The residue was triturated with MTBE/etoac=5/1 (3 mL), the resulting precipitate collected by filtration, washed with MTBE (3 mL) and air dried to give the title compound (680 mg, 99%). LCMS (ESI) m/z:546.3[ M+H ]] + 。
Preparation of compound 10 of scheme 4. Synthesis of allyl (2S, 4R) -4- (((((2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) ethoxy) (hydroxy) phosphoryl) oxy) -2- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidine-1-carboxylate
Figure BDA0004113777320001411
To (2S, 4 r) -4- ((hydroxy (1H-imidazol-1-yl) phosphoryl) oxy) -2- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidine-1-carboxylic acid allyl ester (600 mg,1.1 mmol) and (9H-fluoren-9-yl) methyl (2- (phosphonooxy) ethyl) carbamate (compound 12 in scheme 4 above); description of preparation methods see J.Org.Chem.2007,72,3116.) (400 mg,1.1 mmol) in N, N-dimethylformamide (13 mL) at 23℃Et with 1M zinc chloride was added 2 O (5.5 mL,5.5 mmol). The reaction mixture was stirred at 23 ℃ for 12 hours and then concentrated. The residue was purified by flash chromatography on silica gel (0-30% methanol (3% NH) 3 ·H 2 DCM of O)) to give the title compound as a yellow solid (340 mg, 37%). LCMS (ESI) m/z:814.3[ M+H ]] + 。
Compound 15, scheme 4: synthesis of (9H-fluoren-9-yl) methyl (2- ((hydroxy (((3R, 5S) -5- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) phosphoryl) oxy) ethyl) carbamate
Figure BDA0004113777320001412
Pd (PPh) was added to a 23℃solution of allyl (2S, 4R) -4- (((((2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) ethoxy) (hydroxy) phosphoryl) oxy) -2- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidine-1-carboxylate (340 mg,0.40 mmol) and 1, 3-dimethylpyrimidine-2, 4,6 (1H, 3H, 5H) -trione (316 mg,2.0 mmol) in methylene chloride (5 mL) and methanol (5 mL) at 23 ℃ 3 ) 4 (94 mg,0.08 mmol). The reaction mixture was stirred under nitrogen at 23 ℃ for 2 hours and then concentrated. The residue was purified by preparative HPLC (chromatographic conditions as follows: column: YMC Triart C18. Times.25 mM. Times.5 um; mobile phase: 20-50% water (10 mM NH) 4 HCO 3 ) -ACN; the title compound (202 mg, 66%) was obtained as a white solid on a detector, UV 254 nm. LCMS (ESI) m/z:757.4[ M+H ]] + 。
Compound 16, scheme 4: synthesis of (9H-fluoren-9-yl) methyl (2- (((((((3R, 5S) -1- ((2R) -2- (3- ((3R) -4- (2- ((4- (3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazin-1-yl) ethoxy) isoxazol-5-yl) -3-methylbutanoyl) -5- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) (hydroxy) phosphoryl) oxy) ethyl) carbamate
Figure BDA0004113777320001421
At 23 ℃, a solution containing (2R) -2- (3- (2- ((3R) -4- (2- ((4- (3- (3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl)) 3, 8-diazabicyclo [ 3.2.1)]Octane-8-yl) pyridin-2-yloxy) ethyl) -3-methylpiperazin-1-yl) ethoxy) -3-methylbutanoic acid (308 mg,0.42 mmol), HATU (201 mg,0.53 mmol) and (9H-fluoren-9-yl) methyl (2- ((hydroxy (((3 r, 5S) -5- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-3-yloxy) phosphoryl) oxy) ethyl) carbamate (80 mg,0.11 mmol) anhydrous N, N-dimethylformamide (2 mL) were stirred for 20 minutes. Then N, N-diisopropylethyl Amine (1 mL,0.63 mmol) and the resulting mixture was stirred at 23℃for 2 days. The mixture was purified directly by preparative HPLC, using the following conditions: column: phenomenex Gemini-NX 150 x 30mm x 5um; mobile phase: 24-51% water (0.05% NH) 3 ·H 2 O) -ACN to give the title compound (60 mg, 39%) as a white solid. LCMS (ESI) m/z:1467.7[ M+H ]] + 。
Compound 17, scheme 4: synthesis of [ 2-aminoethoxy (hydroxy) phosphoryl ] [ 3R, 5S) -1- [ (2R) -2- [3- [2- [ (3R) -4- [2- [ [4- [3- [ 3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl ] -3, 8-diazabicyclo [3.2.1] oct-8-yl ] -2-pyridinyl ] oxy ] ethyl ] -3-methyl-piperazin-1-yl ] ethoxy ] isoxazol-5-yl ] -3-methyl-butyryl ] -5- [ (1S) -1- [4- (4-methylthiazol-5-yl) phenyl ] ethyl ] carbamoyl ] pyrrolidin-3-yl ] hydrogen phosphate
Figure BDA0004113777320001431
(9H-fluoren-9-yl) -methyl-containing (2- ((((((3R, 5S) -1- ((2R) -2- ((3R) -4- (2- ((4- (3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl)) 3, 8-diazabicyclo [ 3.2.1)]Octane-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazin-1-yl) ethoxy) isoxazol-5-yl) -3-methylbutanoyl) -5- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) pyrrolidin-3-yl) oxy) (hydroxy) phosphoryl) oxy) ethyl) carbamate (20 mg,0.01 mmol) and piperidine (2 mg,0.01 mmol) in anhydrous N, N-dimethylformamide (0.5 mL) were stirred at 23 ℃ for 2 hours. The mixture was then purified by preparative HPLC (Phenomenex Gemini-NX 150 x 30mm x 5um,20-50% water (0.05% nh 3 ·H 2 O) -ACN) was purified directly to give the title compound (18 mg, 94%) as a white solid. 1 H NMR(400MHz,DMSO-d 6 ):δ9.01-8.91(m,1H),7.93-7.89(m,1H),7.79-7.75(m,1H),7.48(s,1H),7.43-7.28(m,5H),7.23-7.19(m,2H),6.88-6.80(m,3H),6.54-6.52(m,1H),6.14-6.10(m,2H),5.97(s,2H),4.89-4.86(m,2H),4.52-4.35(m,3H),4.26-4.21(m,5H),4.08-3.73(m,5H),3.06-2.98(m,4H),2.95-2.91(m,3H),2.89-2.84(m,6H),2.69-2.66(m,4H),2.46-2.41(m,6H),2.36-2.31(m,2H),2.17-2.15(m,3H),1.98-1.95(m,3H),1.89-1.84(m,2H),1.55-1.51(m,9H),1.39-1.31(m,3H),1.26-1.21(m,3H),1.03-0.92(m,8H),0.80-0.71(m,5H)；LCMS(ESI)m/z：1245.6[M+H] + 。
L1-CIDE-BRM1-4: synthesis of [ (3R, 5S) -1- [2- [3- [2- [ (3R) -4- [2- [ [4- [3- [ 3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl ] -3, 8-diazabicyclo [3.2.1] oct-8-yl ] -2-pyridinyl ] oxy ] ethyl ] -3-methyl-piperazin-1-yl ] ethoxy ] isoxazol-5-yl ] -3-methyl-butyryl ] -5- [ [ (1S) -1- [4- (4-methylthiazol-5-yl) phenyl ] ethyl ] carbamoyl ] pyrrolidin-3-yl ] [2- [6- (2, 5-dioxopyrrol-1-yl) hexanoylamino ] ethoxy-hydroxy-phosphoryl ] hydrogen phosphate
Figure BDA0004113777320001441
To phosphorus containing [ 2-aminoethoxy (hydroxy) phosphoryl groups][ (3R, 5S) -1- [2- [3- [2- [ (3R) -4- [2- [ [4- [3- [ 3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl ]]-3, 8-diazabicyclo [3.2.1]Octane-8-yl]-2-pyridyl group]Oxy group]Ethyl group]-3-methyl-piperazin-1-yl]Ethoxy group]Isoxazol-5-yl]-3-methyl-butyryl]-5- [ [ (1S) -1- [4- (4-methylthiazol-5-yl) phenyl]Ethyl group]Carbamoyl radicals]Pyrrolidin-3-yl]To a solution of hydrogen phosphate (50 mg,0.04 mmol) in N, N-dimethylformamide (2 mL) at 23deg.C was added 1- (6- (2, 5-dioxopyrrolidin-1-yl) -6-oxohexyl) -1H-pyrrole-2, 5-dione (24 mg,0.08 mmol) and N, N-diisopropylethylamine (0.01 mL,0.08 mmol). The mixture was stirred at 23 ℃ for 1 hour, then purified by preparative HPLC (Phenomenex Gemini-NX 80 x 30mM x 3umto,17-47% water (10 mM NH 4 HCO 3 ) ACN) was purified directly to give the title compound (7.9 mg, 14%) as a white solid. 1 H NMR(400MHz,DMSO-d 6 ):δ8.98-8.95(m,1H),8.60-8.41(m,1H),7.94-7.90(m,1H),7.79-7.75(m,1H),7.48(s,1H),7.45-7.30(m,4H),7.23-7.20(m,1H),6.99-6.94(m,2H),6.88-6.80(m,2H),6.54-6.50(m,1H),6.14-6.10(m,1H),5.98-5.96(m,2H),4.89-4.86(m,2H),4.54-4.35(m,3H),4.26-4.21(m,4H),3.95-3.71(m,5H),3.24-3.21(m,1H),3.04-2.98(m,3H),2.82-2.75(m,1H),2.69-2.64(m,1H),2.45-2.42(m,5H),2.36-2.31(m,1H),2.18-2.15(m,2H),2.07-1.92(m,5H),1.79(s,12H),1.46-1.44(m,5H),1.38-1.35(m,3H),1.26-1.22(m,3H),1.16-1.14(m,2H),0.99-0.95(m,6H),0.89
-0.76(m,4H)；LCMS(ESI)m/z：1438.8[M+H] + 。
Synthesis example 5
Synthesis of L1-CIDE-BRM1-5
L1-CIDE-BRM1-2 is synthesized by the following scheme (scheme 5):
Figure BDA0004113777320001451
synthesis of (S) -N- (1- (4-bromophenyl) ethyl) acetamide
Figure BDA0004113777320001452
Acetyl chloride (2.35 g,30 mmol) was added dropwise to a solution containing (S) -1- (4-bromophenyl) ethylamine (5.0 g,25 mmol) and Et at 0deg.C 3 N (3.8 g,37.49 mmol) in THF (50 mL). The reaction was stirred at this temperature for 2.5 hours and then partitioned between EtOAc (50 mL. Times.3) and saturated NaCl solution (50 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography on silica gel (petroleum ether with 0-50% EtOAc) to give the title compound (4.0 g, 66%) as a white solid. LCMS (ESI) m/z:241.8[ M+H ]] + 。
Synthesis of (S) -N- (1- (4-cyanophenyl) ethyl) acetamide
Figure BDA0004113777320001461
A mixture of copper (I) cyanide (1.78 g,20 mmol) and (S) -N- (1- (4-bromophenyl) ethyl) acetamide (4.0 g,16.5 mmol) in N, N-dimethylformamide (40 mL) was refluxed for 24 hours. After cooling to 23 ℃, the mixture was filtered and the filtrate was concentrated. The residue was added to saturated NaHCO at 23 ℃ 3 Aqueous solution (80 mL) and stirred for 10 min. Then saturated aqueous sodium hypochlorite solution was added and stirring was continued for 24 hours at 23 ℃. Mixing The compound was extracted with EtOAc (70 ml×3) and the combined organic layers were washed with water (70 ml×3), dried over sodium sulfate, filtered and concentrated to give the title compound (2.7 g, 87%) as a yellow solid. LCMS (ESI) m/z:189.2[ M+H ]] + 。
Synthesis of (S) -4- (1-aminoethyl) benzonitrile hydrochloride
Figure BDA0004113777320001462
A solution of (S) -N- (1- (4-cyanophenyl) ethyl) acetamide (2.4 g,12.8 mmol) and aqueous 2M HCl (20 mL,12.8 mmol) was stirred at 100deg.C for 24 hours. The reaction solution was cooled to 23 ℃ and then concentrated to give the title compound (1.8 g, 97%) as a yellow solid. LCMS (ESI) m/z:147.1[ M+H ]] + 。
Compound 4, scheme 5: synthesis of (2S, 4R) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester
Figure BDA0004113777320001463
To a mixture of (S) -4- (1-aminoethyl) benzonitrile hydrochloride (1.6 g,11 mmol) and (2S, 4R) -1- (tert-butoxycarbonyl) -4-hydroxypyrrolidine-2-carboxylic acid (2.8 g,12 mmol) in N, N-dimethylformamide (50 mL) at 23℃were added N, N-diisopropylethylamine (4.3 g,33 mmol) and HATU (1.63 g,12 mmol). The reaction was stirred at 23 ℃ for 16 hours and then concentrated. The residue was purified by flash chromatography on silica gel (50-100% ethyl acetate in methylene chloride) to give the title compound (1.7 g, 43%) as a yellow solid. LCMS (ESI) m/z:360.1[ M+H ] ] + 。
Synthesis of tert-butyl (2S, 4R) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -4- ((4-nitrobenzoyl) oxy) pyrrolidine-1-carboxylate
Figure BDA0004113777320001471
To a mixture of 4-nitrophenyl chloroformate (1.68 g,8.3 mmol) and tert-butyl (2S, 4R) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -4-hydroxypyrrolidine-1-carboxylate (3.0 g,7.0 mmol) in dry dichloromethane (80 mL) at 23℃was added 2, 6-lutidine (1.1 g,10.43 mmol). The reaction mixture was stirred at 23 ℃ for 18 hours, and then concentrated to give the title compound (437 mg, 99.8% yield) as a yellow solid. LCMS (ESI) m/z:597.2[ M+H ]] + . Compound 6, scheme 5: synthesis of tert-butyl (2S, 4R) -4- (((1- (4- ((S) -2- (1- ((allyloxy) carbonyl) cyclobutanecarboxamido) -5-ureidovaleramido) phenyl) -2- (4-methylpiperazin-1-yl) -2-oxoethoxy) carbonyl) oxy) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) pyrrolidine-1-carboxylate
Figure BDA0004113777320001473
To a mixture of tert-butyl (2S, 4 r) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -4- ((4-nitrobenzoyl) oxy) pyrrolidine-1-carboxylate (433 mg,0.83 mmol) and 1- (((2S) -1- ((4- (1-hydroxy-2- (4-methylpiperazin-1-yl) -2-oxoethyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylate (334 mg,0.58 mmol) in anhydrous N, N-dimethylformamide (10 mL) at 23 ℃. The reaction mixture was stirred at 40 ℃ for 18 hours, then cooled to 23 ℃ and filtered. The filtrate was directly purified by preparative HPLC (Xtimate C18 150 x 40mm x 5 um/water (0.225% fa) -ACN, 18-48%) to give the title compound (65 mg, 8%) as a yellow solid. LCMS (ESI) m/z:958.6[ M+H ] ] + 。
Compound 7, scheme 5: synthesis of 1- (((2S) -1- ((4- (1- ((((((3R, 5S) -1- (tert-butoxycarbonyl) -5- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) carbonyl) oxy) -2- (4-methylpiperazin-1-yl) -2-oxoethyl) phenyl) amino) -1-oxo-5-ureidopent-2-yl) carbamoyl) cyclobutanecarboxylic acid
Figure BDA0004113777320001482
To a solution of tert-butyl (2S, 4R) -4- (((1- (4- ((S) -2- (1- ((allyloxy) carbonyl) cyclobutanecarboxamido) -5-ureidovaleramido) phenyl) -2- (4-methylpiperazin-1-yl) -2-oxoethoxy) carbonyl) oxy) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) pyrrolidine-1-carboxylate (65 mg,0.07 mmol) and 1, 3-dimethylpyrimidine-2, 4,6 (1H, 3H, 5H) -trione (53 mg,0.34 mmol) in dichloromethane (3 mL) and methanol (3 mL) at 23℃was added Pd (PPh) 3 ) 4 (16 mg,0.01 mmol). The reaction mixture was stirred under nitrogen at 23 ℃ for 10 hours and then concentrated. The residue was purified by preparative HPLC (chromatographic conditions as follows: column Phenomenex Gemini-NX 80X 30mM 3um, mobile phase (24-50%) water (10 mM NH) 4 HCO 3 ) ACN to give the title compound as a red solid (40 mg, 64%). LCMS (ESI) m/z:918.6[ M+H ]] + 。
Compound 9, scheme 5: synthesis of (2S, 4R) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -4- (((1- (4- ((S) -2- (1- ((5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) carbamoyl) cyclobutanecarboxamide) phenyl) -5-ureidovaleryl) phenyl) -2- (4-methylpiperazin-1-yl) -2-oxoethoxy) carbonyl) oxy) pyrrolidine-1-carboxylic acid tert-butyl ester
Figure BDA0004113777320001491
To a mixture of 1- (((2S) -1- ((4- (1- ((((((3 r, 5S) -1- (tert-butoxycarbonyl) -5- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) carbonyl) oxy) -2- (4-methylpiperazin-1-yl) -2-oxoethyl) phenyl) amino) -1-oxo-5-ureidopent-2-yl) carbamoyl) cyclobutanecarboxylic acid (40 mg,0.04 mmol) and 1- (5-aminopentyl) -1H-pyrrole-2, 5-dione (10 mg,0.05 mmol) in N, N-dimethylformamide (4 mL) was added N, N-diisopropylethylamine (0.02 mL, 0.05 mmol) and HATU (20 mg,0.05 mmol). The reaction mixture was stirred at 23 ℃ for 16 hours and then concentrated. The residue was purified by preparative HPLC (Boston Green ODS 150 x 30mm x 5um, water (0.075% tfa) -ACN,20% -50%) to give a blue colorThe title compound (31 mg, 65%) was obtained as a solid. LCMS (ESI) m/z:1082.7[ M+H ]] + 。
Synthesis of 2, 2-trifluoroacetate ester of (3R, 5S) -5- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) pyrrolidin-3-yl (1- (4- ((S) -2- (1- ((5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) carbamoyl) cyclobutanecarboxamido) -5-ureidovaleryl) phenyl) -2- (4-methylpiperazin-1-yl) -2-oxoethyl) carbonate
Figure BDA0004113777320001492
A HFIP (3 mL) solution containing 5% TFA of (2S, 4R) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -4- (((1- (4- ((S) -2- (1- ((5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) carbamoyl) cyclobutanecarboxamido) -5-ureidovaleryl) phenyl) -2- (4-methylpiperazin-1-yl) -2-oxoethoxy) carbonyl) oxy) pyrrolidine-1-carboxylic acid tert-butyl ester (30.5 mg,0.03 mmol) was stirred at 23℃for 1.5 hours. The reaction mixture was then concentrated to give the title compound (28 mg, 99%) as a powder oil. LCMS (ESI) m/z:982.7[ M+H ] ] + 。
L1-CIDE-BRM-1-5: synthesis of (3R, 5S) -1- (2- (3- (4- (trans-3- ((4- (3- (3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) cyclobutoxy) piperidin-1-yl) methyl) piperidin-1-yl) -isoxazol-5-yl) -3-methylbutanoyl) -5- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) pyrrolidin-3-yl (1- (4- ((S) -2- (1- ((5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) carbamoyl) cyclobutanecarboxamido) -5-ureidovaleryl) phenyl) -2- (4-methylpiperazin-1-yl) -2-oxoethyl) carbonate
Figure BDA0004113777320001501
At 23 ℃, to a (3 r, 5S) -5- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) containing pyrrolidin-3-yl (1- (4- ((S) -2- (1- ((5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) phenyl) carbamoyl)Cyclobutanecarboxamido) -5-ureidovaleramido) phenyl) -2- (4-methylpiperazin-1-yl) -2-oxoethyl) carbonate 2, 2-trifluoroacetate (28 mg,0.03 mmol) and 2- (3- (4- ((4- (trans-3- ((4- (3- (3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]Octan-8-yl) pyridin-2-yl) oxy) cyclobutoxy) piperidin-1-yl) methyl piperidin-1-yl) isoxazol-5-yl) -3-methylbutanoic acid (27 mg,0.03 mmol) in DMF (3 mL) was added N, N-diisopropylethylamine (0.01 mL,0.08 mmol) and HATU (13 mg,0.03 mmol). The mixture was stirred at 23 ℃ for 4 hours and then concentrated. The residue was purified by preparative HPLC (chromatographic conditions as follows: column Phenomenex Gemini-NX 80X 30mM 3um; mobile phase (18-36%) water (10 mM NH) 4 HCO 3 ) ACN to give the title compound (23 mg, 31%) as a white solid. 1 H NMR(400MHz,CD 3 OD):δ7.80-7.58(m,6H),7.57-7.34(m,5H),7.24-7.21(m,1H),6.96-6.84(m,2H),6.80-6.73(m,1H),6.56-6.54(m,1H),6.33-6.20(m,1H),6.17-6.03(m,1H),5.27-5.19(m,1H),5.14-5.12(m,1H),4.62-4.60(m,5H),4.54-4.51(m,3H),4.42-4.39(m,1H),4.17-3.86(m,2H),3.84-3.76(m,1H),3.74-3.64(m,3H),3.63-3.50(m,4H),3.48-3.43(m,3H),3.38-3.35(m,2H),3.25-3.22(m,2H),3.26-3.18(m,1H),3.17-3.00(m,4H),2.86-2.83(m,2H),2.59-2.55(m,6H),2.48-2.35(m,7H),2.29-2.12(m,8H),2.09-1.86(m,8H),1.85-1.68(m,1H),1.79-1.75(m,5H),1.67-1.52(m,7H),1.51-1.43(m,2H),1.41-1.23(m,9H),1.10-0.94(m,3H),0.93-0.81(m,3H)；LCMS(ESI)m/z：1772.9[M+H] + 。
Synthesis example 6
Synthesis of L1-CIDE-BRM1-6
L1-CIDE-BRM1-6 is synthesized by the following scheme (scheme 6):
Figure BDA0004113777320001511
synthesis of (2S, 4R) -N- ((S) -1- (4-cyanophenyl) ethyl) -4-hydroxypyrrolidine-2-carboxamide hydrochloride
Figure BDA0004113777320001512
To a solution of (2S, 4 r) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester (see compound 4 in scheme 5 above) (2.0 g,5.56 mmol) in ethyl acetate (5 mL) at 23 ℃ C. Was added 4M HCl (10 mL; prepared by bubbling dry HCl gas in anhydrous EtOAc). The reaction mixture was stirred at 23 ℃ for 16 hours, then concentrated to give the title compound (1.4 g, 97%) as a white solid.
Compound 4, scheme 6: synthesis of (2S, 4R) -N- ((S) -1- (4-cyanophenyl) ethyl) -1- (2- (3- (4- (dimethoxymethyl) piperidin-1-yl) isoxazol-5-yl) -3-methylbutanoyl) -4-hydroxypyrrolidine-2-carboxamide
Figure BDA0004113777320001521
To a 23℃solution of (2S, 4R) -N- ((S) -1- (4-cyanophenyl) ethyl) -4-hydroxypyrrolidine-2-carboxamide hydrochloride (900 mg,3.47 mmol) and 2- (3- (4- (dimethoxymethyl) piperidin-1-yl) isoxazol-5-yl) -3-methylbutanoic acid (see compound 3 in scheme 6 above; see synthetic route disclosed on page 450 of US 2020/0038378, incorporated herein by reference in its entirety) (1.4 g,3.43 mmol) in dry N, N-dimethylformamide (20 mL) was added DIEA (1.71 mL,10.29 mmol) and HATU (2.0 g,5.15 mmol). The mixture was stirred at 23 ℃ for 2 hours, then partitioned between water (100 mL) and ethyl acetate (100 mL x 3). The combined organic layers were washed with brine (50 ml×2), dried over sodium sulfate, filtered, and then concentrated. The residue was purified by flash chromatography on silica gel (petroleum ether containing 0-100% ethyl acetate) to give the title compound (2.0 g, 98%) as a colourless oil. LCMS (ESI) m/z:568.3[ M+H ] ] + 。
Compound 5, scheme 6: synthesis of (2S, 4R) -N- ((S) -1- (4-cyanophenyl) ethyl) -1- ((R) -2- (3- (4- (dimethoxymethyl) piperidin-1-yl) isoxazol-5-yl) -3-methylbutanoyl) -4-hydroxypyrrolidine-2-carboxamide
Figure BDA0004113777320001531
By chiral SFC (DAICEL CHIRALPAK OD (250 mm. Times.30 mm,10 um); (20%) 0.1% NH 3 H 2 O, etOH) isolation of (2S, 4R) -N- ((S) -1- (4-cyanophenyl) ethyl) -1- (2- (3- (4- (dimethoxymethyl) piperidin-1-yl) isoxazol-5-yl) -3-methylbutyryl) -4-hydroxypyrrolidine-2-carboxamide (2 g,3.52 mmol) gives a first peak (2S, 4R) -N- ((S) -1- (4-cyanophenyl) ethyl) -1- ((S) -2- (3- (4- (dimethoxymethyl) piperidin-1-yl) isoxazol-5-yl) -3-methylbutyryl) -4-hydroxypyrrolidine-2-carboxamide (400 mg, 20%) and a second peak (2S, 4R) -N- ((S) -1- (4-cyanophenyl) ethyl) -1- ((R) -2- (3- (4- (dimethoxymethyl) piperidin-1-yl) isoxazol-5-yl) -3-methylbutyryl) -4-hydroxypyrrolidine-2-carboxamide (mg, 35%) as a white solid. 1 H NMR(400MHz,DMSO-d 6 ,):δ8.49(d,J＝7.2Hz,1H),7.82-7.76(m,2H),7.50-7.44(m,2H),6.11(s,1H),5.13(d,J＝3.6Hz,1H),4.93-4.89(m,1H),4.36-4.31(m,1H),4.27-4.25(s,1H),4.07(d,J＝7.6Hz,1H),3.72-3.53(m,4H),3.45-3.40(m,1H),3.26(s,6H),2.76-2.67(m,2H),2.25-2.16(m,1H),2.08-1.96(m,1H),1.79-1.61(m,4H),1.44-1.33(m,3H),1.30-1.19(m,2H),0.98-0.89(m,3H),0.84-0.74(m,3H)。
Compound 8, scheme 6: synthesis of S- (3- (((((3R, 5S) -5- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -1- ((R) -2- (3- (4- (dimethoxymethyl) piperidin-1-yl) isoxazol-5-yl) -3-methylbutanoyl) pyrrolidin-3-yl) oxy) carbonyl) oxy) butan-2-yl) methylthiosulfonate
Figure BDA0004113777320001532
To a solution containing (2S, 4R) -N- ((S) -1- (4-cyanophenyl) ethyl) -1- ((R) -2- (3- (4- (dimethoxymethyl) piperidin-1-yl) isoxazol-5-yl) -3-methylbutanoyl) -4-hydroxypyrrolidine-2-carboxamide (300 mg,0.53 mmol) and
Figure BDA0004113777320001541
s- (3- ((chlorocarbonyl) oxy) butan-2-yl was slowly added to a 23℃mixture of dry dichloromethane (3 mL) of MS (100 mg)) Methylthiosulfonic acid salts (see Compound 7 in scheme 6 above or Compound 2 in scheme 1 above) ((399mg, 1.59 mmol) in dichloromethane (2 mL) and Et-containing salts) 3 N (214 mg,2.11 mmol) in dry dichloromethane (3 mL). The reaction was stirred at 23 ℃ for 16 hours and then filtered. The filtrate was concentrated, and the residue was purified by flash chromatography on silica gel (petroleum ether containing 0-70% ethyl acetate) to give the title compound (200 mg, 49%) as a white solid. LCMS (ESI) m/z:778.1[ M+H ]] + 。
Compound 9, scheme 6: synthesis of S- (3- (((((3R, 5S) -5- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -1- ((R) -2- (3- (4-formylpiperidin-1-yl) isoxazol-5-yl) -3-methylbutanoyl) pyrrolidin-3-yl) oxy) carbonyl) oxy) butan-2-yl) methylthiosulfonate
Figure BDA0004113777320001542
To a solution of S- (3- (((((3R, 5S) -5- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -1- ((R) -2- (3- (4- (dimethoxymethyl) piperidin-1-yl) isoxazol-5-yl) -3-methylbutanoyl) pyrrolidin-3-yl) oxy) carbonyl) oxy) butan-2-yl) methylthiosulfonate (100 mg,0.13 mmol) in THF (1 mL) was added formic acid (1 mL) and water (3 mL) at 23 ℃. The mixture was stirred at 50 ℃ for 16 hours, then cooled to 23 ℃ and concentrated to give the title compound (80 mg, 85%) as a yellow oil. LCMS (ESI) m/z:732.0[ M+H ] ] + 。
L1-CIDE-BRM1-6: synthesis of S- (3- (((((3R, 5S) -1- ((2R) -2- (3- ((4- (trans-3- ((4- (3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) cyclobutoxy) piperidin-1-yl) methyl) piperidin-1-yl) isoxazol-5-yl) -3-methylbutyl) -5- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) butan-2-yl) methylthiosulfonate
Figure BDA0004113777320001551
To a polypeptide containing S- (3- ((((3)R, 5S) -5- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -1- ((R) -2- (3- (4-formylpiperidin-1-yl) -3-methylbutanoyl) pyrrolidin-3-yl) oxy) carbonyl) butan-2-yl) methylthiosulfonate (80 mg,0.11 mmol) and 2- (6-amino-5- (8- (2- (3- (piperidin-4-yloxy) cyclobutoxy) pyridin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]Octan-3-yl) pyridazin-3-yl phenol hydrochloride (see compound 5 in scheme 6 above; see U.S. Pat. No. 2020/0038378, pages 306-307, incorporated herein by reference in its entirety) (60 mg,0.11 mmol) and HOAc (0.2 mL) in dichloromethane (1 mL) and methanol (1 mL) at 23℃with the addition of NaBH (OAc) 3 (232 mg,1.09 mmol). The reaction mixture was stirred at 23 ℃ for 3 hours and then concentrated. The residue was purified by preparative HPLC (Boston Green ODS 150 x 30mm x 5um (water (0.225% fa) -ACN, 15-45%) to give the title compound (35 mg, 26%) as a white solid. After purification by preparative HPLC (FA), the desired product was the FA salt. 1 H NMR(400MHz,DMSO-d 6 ):δ8.55-8.53(m,1H),8.17-8.15(m,1H),7.91(d,J＝7.6Hz,1H),7.81-7.75(m,3H),7.51-7.44(m,3H),7.23-7.20(m,1H),6.89-6.82(m,2H),6.54-6.51(m,1H),6.12(d,J＝8.8Hz,2H),5.97(s,2H),5.26-5.07(m,2H),5.01-4.85(m,2H),4.51-4.46(m,2H),4.38-4.27(m,2H),3.87-3.80(m,2H),3.59-3.56(m,3H),3.54-3.52(m,3H),3.28-3.24(m,1H),3.03-2.99(m,2H),2.76-2.70(m,2H),2.36-2.31(m,2H),2.19-2.09(m,4H),2.01-1.94(m,4H),1.80-1.62(m,5H),1.45-1.32(m,9H),1.27-1.25(m,1H),1.14-1.02(m,2H),0.95-0.89(m,3H),0.88-0.70(m,3H)；LCMS(ESI)m/z：1259.1[M+H] + 。
Synthesis example 7
Synthesis of L1-CIDE-BRM1-7
The scheme is as follows:
Figure BDA0004113777320001561
experiment:
general procedure for preparation of compound 7:
Figure BDA0004113777320001562
to a stirred solution of isobutyraldehyde (2.7 mL,29.6 mmol) in carbon tetrachloride (10 mL) was added dropwise disulfide (1.2 mL,14.8 mmol) at 50deg.C under nitrogen. The reaction was stirred at 30 ℃ under a nitrogen stream for a further 48 hours to remove the liberated hydrogen chloride. TLC (petroleum ether with 25% ethyl acetate, rf=0.5) indicated the reaction was complete. The solution was distilled under vacuum and purified by flash chromatography (eluting with 25% ethyl acetate in petroleum ether) to give 2- [ (1, 1-dimethyl-2-oxo-ethyl) disulfanyl as a colourless oil]2-methyl-propanal (3000 mg, 98.2%). 1 H NMR (400 MHz, chloroform-d): δ=9.09 (s, 2H), 1.37 (s, 12H).
General procedure for preparation of compound 8:
Figure BDA0004113777320001563
to a solution of 2,2' -dithioalkanediylbis (2-methylpropane) (1500.0 mg,7.3 mmol) in tetrahydrofuran (30 mL) was added dropwise methyl magnesium bromide (9.7 mL,29.1 mmol) at 0℃over 5 min. The mixture was stirred at 0℃for 2 hours.
TLC (petroleum ether with 20% ethyl acetate, rf=0.5) indicated the reaction was complete. The mixture was treated with saturated NH 4 Aqueous Cl (10 mL) was quenched and extracted with EtOAc (20 mL. Times.3). The combined organic phases were washed with water (20 mL) and brine (10 mL), dried over Na2SO4, filtered and concentrated to give 3- [ (2-hydroxy-1, 1-dimethyl-propyl) disulfanyl as a yellow oil ]-3-methyl-butan-2-ol (137mg, 79%). 1 H NMR (400 MHz, chloroform-d): δ=3.77-3.74 (m, 1H), 1.31 (s, 6H), 1.26 (d, j=2.4 hz, 3H), 1.19 (d, j=6.4 hz, 3H).
General procedure for preparation of compound 2:
Figure BDA0004113777320001571
to a catalyst containing a 2-methyl-2- [ (5-nitro-2-pyridinyl) dithioalkyl group at 25 ℃C]To a solution of propan-1-ol (5983.8 mg,22.99 mmol) in dichloromethane (50 mL) was added 3- [ (2-hydroxy)-1, 1-dimethyl-propyl) disulfanyl]-3-methyl-butan-2-ol (1370.0 mg,5.75 mmol) and iodine (2917 mg,11.49 mmol). The reaction mixture was stirred at 45 ℃ for 24 hours. TLC (petroleum ether with 33% EtOAc rf=0.4) showed the reaction was complete. The mixture was filtered and the filtrate concentrated in vacuo, then purified by flash chromatography (eluting with 0-50% EtOAc in petroleum ether) to give 3-methyl-3-methylsulfonyl sulfanyl-butan-2-ol as a yellow oil (460 mg, 40.4% yield). 1 HNMR (400 MHz, chloroform-d) delta=4.14-4.09 (m, 1H), 3.42 (s, 3H), 1.68 (s, 3H), 1.45 (s, 3H), 1.29 (d, j=6.4 hz, 3H).
General procedure for preparation of compound 3:
Figure BDA0004113777320001572
to a solution of triphosgene (112.3 mg,0.38 mmol) in dichloromethane (2 mL) was added a solution of 3-methyl-3-methylsulfonylthioalkyl-butan-2-ol (150.0 mg,0.76 mmol) and pyridine (239.3 mg,3.03 mmol) in dichloromethane (2 mL) and the reaction was stirred at 25℃for 30 min. The reaction mixture was concentrated to dryness to give a crude product which was used directly in the next step.
To the above crude product in anhydrous dichloromethane (2 mL) solution was added
Figure BDA0004113777320001573
MS (100 mg) then a solution of triethylamine (32.9 mg,0.33 mmol) and compound 1 (50.0 mg,0.08 mmol) in dry dichloromethane (2 mL) was slowly added at 20deg.C and stirred for an additional 16 h. The residue was concentrated and purified by silica gel flash chromatography (eluting with petroleum ether containing 0-70% ethyl acetate) to give compound 3 (64 mg, 93.8%) as a white solid. LCMS (5-95, ab,1.5 min): rt=0.974 min, m/z=839.3 [ m+h ]] + 。
General procedure for preparation of compound 4:
Figure BDA0004113777320001581
a solution of compound 3 (50.0 mg,0.06 mmol) in formic acid (2 mL) and water (2 mL) was stirred at 50℃for 1 hour. The reaction mixture was concentrated to give compound 4 (45 mg, 98.7%) as a yellow solid, which was used directly in the next step.
LCMS(5-95,AB,1.5min):R T ＝0.848min,m/z＝765.2[M+H] + 。
General procedure for preparation of L1-CIDE-BRM 1-7:
Figure BDA0004113777320001582
to a solution of compound 4 (45.0 mg,0.06 mmol) in dichloromethane (2 mL) was added compound 5 (33.1 mg,0.06 mmol) and sodium triacetoxyborohydride (249.4 mg,1.18 mmol). The reaction mixture was stirred at 20℃for 3 hours. The mixture was concentrated and purified by TLC (8% meoh in DCM) to give L1-CIDE-BRM1-7 (15.0 mg, 19.4%) as a white solid.
1 H NMR (400 MHz, methanol-d 4): delta = 8.88 (s, 1H), 7.81-7.75 (m, 2H), 7.49-7.41 (m, 5H), 7.24-7.20 (m, 1H), 6.92-6.87 (m, 2H), 6.57 (d, J = 4.0hz, 1H), 6.22 (d, J = 2.0hz, 1H), 6.01 (d, J = 3.2hz, 1H), 5.28-5.24 (m, 1H), 5.05-5.02 (m, 1H), 4.61 (s, 2H), 4.53-4.49 (m, 3H), 4.36-4.31 (m, 4H), 4.04-3.90 (m, 2H), 3.67-3.65 (m, 1H), 3.46-3.43 (m, 1H), 3.39 (s, 3H), 3.16-3.03 (m, 2H), 2.95-2H), 4.52-2.52 (m, 2H), 4.52-4.33 (m, 2H), 4.46-3.33 (m, 2H), 4.52 (m, 2H), 4.33-4.33 (m, 2H), 2.4.52 (2H), 2.32 (m, 2H).
LCMS(5-95,AB,1.5min):R T ＝0.829min,m/z＝633.5[M/2+H] + 。HRMS(5-95AB):m/z＝1265.5126[M+H] + 。
Synthesis example 8
Synthesis of L1-CIDE-BRM1-8
The scheme is as follows:
Figure BDA0004113777320001591
experiment:
general procedure for preparation of compound 2:
Figure BDA0004113777320001592
a solution of Compound 1 (120.00 mg,0.20 mmol) in dichloromethane (2.00 mL) and trifluoroacetic acid (0.40 mL) was stirred at 25℃for 1 hour. TLC (10% MeOH in DCM, rf=0.6) showed that most of the starting material had been consumed and a new spot formed. Water (3.00 mL) was then added to the mixture and the mixture was quenched with saturated NaHCO 3 (8.00 mL) was adjusted to ph=9. The mixture was then extracted with dichloromethane (15 ml×3). The organic layer was concentrated to give crude compound 2 (90.00 mg, 85.3%) as a white solid. LCMS (5-95, AB,1.5 min): R T ＝0.789min,m/z＝541.3[M+H] + 。
General procedure for preparation of compound 4:
Figure BDA0004113777320001593
to a solution of compound 3 (75.50 mg,0.14 mmol) and compound 2 (90.00 mg,0.14 mmol) in methanol (5.00 mL) and methylene chloride (5.00 mL) were added sodium cyanoborohydride (12.9 mg,0.20 mmol) and sodium acetate (16.80 mg,0.20 mmol). The mixture was stirred at 25℃for 12 hours. TLC (DCM with 12% MeOH, rf=0.6) showed that most of the starting material had been consumed and a new spot formed. The mixture was filtered and the filtrate was concentrated and purified by preparative TLC (12% MeOH in DCM) to give compound 4 (40.00 mg, 28.1%) as a white solid. LCMS (5-95, AB,1.5 min): R T ＝0.755min,m/z＝1041.2[M+H] + 。
General procedure for preparation of compound 6:
Figure BDA0004113777320001601
to a mixture of triphosgene (18.3 mg,0.062 mmol) and 4A molecular sieve in dichloromethane (2.0 mL) was added a solution of 4- (hydroxymethyl) -4-methylsulfonyl sulfanyl-piperidine-1-carboxylic acid tert-butyl ester (20.0 mg,0.062 mmol) and pyridine (0.02 mL,0.184 mmol) in dichloromethane (2.0 mL). The reaction mixture was stirred at 20℃for 30 min. The reaction mixture was concentrated to give a crude product which was used directly in the next step.
To a crude product containing the above
Figure BDA0004113777320001602
To anhydrous dichloromethane (5.00 mL) of MS (100 mg) were added a solution of N, N-diisopropylethylamine (0.02 mL,0.09 mmol) and compound 4 (30.00 mg,0.03 mmol) in anhydrous N, N-dimethylformamide (2.00 mL). The mixture was stirred at 25℃for 16 hours. TLC (DCM with 11% MeOH, rf=0.6) showed that most of the starting material had been consumed and a new spot formed. The mixture was filtered and concentrated to give a crude product which was purified by preparative TLC (11% MeOH in DCM) to give compound 6 (25.00 mg, 62.3%) as a pale yellow solid. LCMS (10-80, AB,7.0 min): R T ＝3.096min,m/z＝697.1[M/2+H] +
General procedure for preparation of compound 7:
Figure BDA0004113777320001603
to a solution of compound 6 (20.00 mg,0.01 mmol) in dichloromethane (1.00 mL) was added trifluoroacetic acid (0.80 mL,10.38 mmol). The mixture was stirred at 25℃for 1 hour. The mixture was concentrated to give crude product 7, which was used directly as TFA salt in the next step.
General procedure for preparation of L1-CIBE-BRM 1-8:
Figure BDA0004113777320001611
to a solution of formaldehyde (4.3 mg,0.14 mmol) and compound 7 (20.20 mg,0.01 mmol) in dichloromethane (2 mL) and methanol (1 mL) was added acetic acid (1.00 mg). The mixture was stirred at 25℃for 30 minutes. Then, sodium triacetoxyborohydride (9.2 mg,0.04 mmol) was added. Will beThe mixture was stirred at 25℃for 1 hour. The mixture was then diluted with DCM (15 mL) and saturated NaHCO 3 (5 mL) was washed and the organic layer was concentrated, then the residue was purified by reverse phase chromatography (aqueous solution containing acetonitrile 14-44/0.225% FA) to give L1-CIDE-BRM1-8 (3.60 mg, 18.2%) as a white solid.
1 H NMR(400MHz,DMSO-d6):δ＝8.99(s,1H),8.49(d,J＝8.0Hz,1H),8.15(s,1H),7.91(d,J＝8.0Hz,1H),7.78(d,J＝6.0Hz,1H),7.49-7.36(m,6H),7.22-7.20(m,1H),6.88-6.85(m,2H),6.54-6.52(m,1H),6.13(d,J＝8.0Hz,2H),5.97-5.94(m,2H),5.25-5.21(m,1H),4.93-4.89(m,1H),4.52-4.43(m,6H),4.26-4.21(m,6H),3.87(br s,1H),3.72(d,J＝9.2Hz,1H),3.52(s,3H),3.02-2.96(m,4H),2.61(br s,4H),2.46–2.33(m,10H),2.20-2.14(m,6H),1.96-1.89(m,10H),1.38(d,J＝7.2Hz,3H),0.99-0.95(m,6H),0.81(d,J＝6.4Hz,3H)。LCMS(5-95,AB,1.5min):R T ＝0.781min,m/z＝1306.5[M+H] + 。
General procedure for preparation of compound 9:
Figure BDA0004113777320001612
to a mixture of carbon tetrachloride (150 mL) with tert-butyl 4-formyl-1-piperidinecarboxylate (9870.7 mg,46.28 mmol) was added dithio dichloride (1.48 mL,18.51 mmol) and the mixture stirred at 55 ℃ for 16 hours, TLC (10% methanol in dichloromethane, rf=0.5) indicated completion of the reaction. The mixture was filtered and the organic layer was concentrated in vacuo. To the residue was added water (30 mL) and extracted with dichloromethane (3×50 mL), the organic layers were combined and taken up with Na 2 SO4 was dried, filtered and concentrated to give a crude product by preparative TLC (10% methanol in dichloromethane, R f =0.5) to give 4- [ (1-tert-butoxycarbonyl-4-formyl-4-piperidinyl) disulfanyl as a white solid]-4-formyl-piperidine-1-carboxylic acid tert-butyl ester (5.5 g, 60.8%). 1 H NMR (400 MHz, chloroform-d) delta=9.06 (s, 2H), 3.73 (br s, 4H), 3.18-3.11 (m, 4H), 2.07-2.02 (m, 4H), 1.74-1.69 (m, 4H), 1.46 (s, 18H).
General procedure for preparation of compound 10:
Figure BDA0004113777320001621
to 4- [ (1-tert-butoxycarbonyl-4-formyl-4-piperidinyl) disulfanyl group]To a mixture of tert-butyl-4-formyl-piperidine-1-carboxylate (3000.0 mg,6.14 mmol) in methanol (40 mL) was added sodium borohydride (696.7 mg,18.42 mmol), the mixture was stirred at 25℃for 1 h, TLC (10% methanol in dichloromethane, rf=0.5) showed a new spot, the reaction was stopped by adding water (30 mL) and the resulting mixture was extracted with dichloromethane (3X 30 mL), the organic layers were combined and the reaction was quenched with Na 2 SO4 is dried, filtered and concentrated to give a crude product which is purified by silica gel chromatography (eluting with 0-3% methanol in methylene chloride) to give 4- [ 1-tert-butoxycarbonyl-4- (hydroxymethyl) -4-piperidinyl as a white solid]Disulfanyl group]-4- (hydroxymethyl) piperidine-1-carboxylic acid tert-butyl ester (3000 mg, 99%). 1 H NMR (400 MHz, chloroform-d) delta=3.75-3.72 (m, 4H), 3.59 (s, 4H), 3.32-3.26 (m, 4H), 1.75-1.67 (m, 8H), 1.46 (s, 18H).
General procedure for preparation of compound 11:
Figure BDA0004113777320001631
to a suspension of lithium aluminum hydride (1232.4 mg,32.47 mmol) in tetrahydrofuran (40 mL) was added dropwise a solution containing 4- [ [ 1-tert-butoxycarbonyl-4- (hydroxymethyl) -4-piperidinyl group)]Disulfanyl group]A solution of tert-butyl 4- (hydroxymethyl) piperidine-1-carboxylate (3200.0 mg,6.49 mmol) in tetrahydrofuran (40 mL). The resulting mixture was stirred under nitrogen at 25 ℃ for 2 hours. NH for reaction 4 Aqueous Cl (10 mL) was quenched and extracted with ethyl acetate (30 mL. Times.3). The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo to give crude 4- (hydroxymethyl) -4-thio-piperidine-1-carboxylic acid tert-butyl ester (2700 mg, 100%) which was used directly in the next step. 1 H NMR (400 MHz, chloroform-d) delta=3.96-3.92 (m, 2H), 3.52 (s, 2H) 3.27-3.21 (m, 2H), 1.64-1.61 (m, 4H), 1.47 (s, 9H).
General procedure for preparation of compound 12:
Figure BDA0004113777320001632
to a solution of imidazole (1783.5 mg,26.2 mmol) and tert-butyl 4- (hydroxymethyl) -4-thio-piperidine-1-carboxylate (2700.0 mg,10.92 mmol) in dichloromethane (40 mL) was added dichloromethane (40 mL) containing tert-butyldimethylchlorosilane (2467.8 mg,16.37 mmol). The mixture was stirred continuously at 20℃for 12 hours. TLC (petroleum ether with 20% ethyl acetate, rf=0.58) indicated the reaction was complete. The mixture was then washed with water (20 mL), the organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum, and the crude product (petroleum ether containing 10% ethyl acetate, rf=0.58) was purified using silica gel chromatography to give 4- [ [ tert-butyl (dimethyl) silicon-based ]Oxymethyl group]-4-thio-piperidine-1-carboxylic acid tert-butyl ester (380 mg, 96.3%). 1 H NMR (400 MHz, chloroform): δ=3.96-3.92 (m, 2H), 3.52 (s, 2H), 3.20-3.13 (m, 2H), 1.72-1.65 (m, 2H), 1.52-1.49 (m, 2H), 1.45 (s, 9H), 0.90 (s, 9H), 0.06 (s, 6H).
General procedure for preparation of compound 13:
Figure BDA0004113777320001641
to a solution of methanesulfonyl chloride (2.51 g,21.91 mmol) in dichloromethane (20 mL) under N2 protection was added dropwise a solution containing 4- [ [ tert-butyl (dimethyl) silyl group]Oxymethyl group]A solution of tert-butyl 4-thio-piperidine-1-carboxylate (3.8 g,10.51 mmol) and triethylamine (5.45 mL,42.03 mmol) in dichloromethane (20 mL). The mixture was stirred at 25℃for 2 hours. TLC (petroleum ether with 20% ethyl acetate, rf=0.3) showed a new spot. The reaction was quenched with water (30 mL) and extracted with dichloromethane (30 mL x 3). The organic layer was concentrated and purified by silica gel column (eluting with petroleum ether containing 0-10% ethyl acetate) to give 4- [ [ tert-butyl (dimethyl) silicon-based ] as a white solid]Oxymethyl group]-4-methylsulfonyl sulfanyl-piperidine-1-carboxylic acid tert-butyl ester (1670 mg, 36.1%). 1 H NMR (400 MHz, chloroform): δ=3.94-3.88 (m, 4H), 3.39 (s, 3H), 3.26-3.20 (m, 1H), 1.99-1.95 (m, 2H), 1.86-1.80 (m, 2H), 1.46 (s, 9H), 0.91(s,9H),0.10(s,6H)。LCMS(5-95,AB,1.5min):R T ＝1.126min,m/z＝340.1[M-100+H] + 。
General procedure for preparation of compound 5:
Figure BDA0004113777320001642
To 4- [ [ tert-butyl (dimethyl) silicon-based)]Oxymethyl group]To a solution of tert-butyl 4-methylsulfonyl sulfanyl-piperidine-1-carboxylate (1500.00 mg,3.41 mmol) in tetrahydrofuran (10.0 mL) was added tetrabutylammonium fluoride (5.12 mL,5.12mmol, 1mol/L in THF). The mixture was stirred at 0℃for 20 min. TLC (60% EtOAc in petroleum ether, rf=0.4) showed most of the starting material was consumed. EtOAc (70 mL) was added to the mixture, the organic layer was washed with water (25 mL x 2), brine (25 mL), and the organic layer was concentrated to give a crude product, which was purified by silica gel column flash chromatography (eluting with 0-60% EtOAc in petroleum ether) to give tert-butyl 4- (hydroxymethyl) -4-methylsulfonylthanyl-piperidine-1-carboxylate (670.00 mg, 60.4%) as a colourless oil. 1 H NMR (400 MHz, chloroform-d) delta=3.96 (s, 2H), 3.89-3.85 (m, 2H), 3.43 (s, 3H), 3.31-3.28 (m, 2H), 2.12-2.08 (m, 2H), 1.82-1.76 (m, 2H), 1.47 (s, 9H).
Synthesis example 9
Synthesis of L1-CIDE-BRM1-9
Figure BDA0004113777320001651
Step 1: (3R) -4- (2- ((4- (3-amino-6- (2- (((di-tert-butoxyphosphoryl) oxy) methoxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester
Figure BDA0004113777320001652
To a solution of tert-butyl (3R) -4- (2- ((4- (3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazine-1-carboxylate (300 mg,0.49 mmol) in 1-methylpyrrolidin-2-one (8.0 mL) was added cesium carbonate (0.32 g,0.97 mmol) and di-tert-butylchloromethyl phosphate (0.19 g,0.73 mmol). The reaction mixture was stirred at 45℃for 12 hours. The reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with brine (10 ml x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (silica gel, 100-200 mesh, dichloromethane containing 0-2% methanol) to give the title compound (200 mg, yield 49%) as a yellow oil.
LCMS(ESI)m/z：839.3[M+H] + 。
Step 2: (2- (6-amino-5- (8- (2- (2- ((R) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) methyl phosphate monobasic salt
Figure BDA0004113777320001661
To a solution of tert-butyl (3R) -4- (2- ((4- (3-amino-6- (2- (((di-tert-butoxyphosphoryl) oxy) methoxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazine-1-carboxylate (200 mg,0.24 mmol) was added hexafluoroisopropanol (5.0 mL) containing 5% trifluoroacetic acid. The reaction mixture was stirred at 20℃for 3 hours. The reaction was concentrated and purified by Column Boston Green ODS, 150 x 30mm x 5um (over water (0.225% formic acid) -acetonitrile (5% -35%)) to give the title compound as a yellow solid (100 mg, 56.6% yield).
LCMS(ESI)m/z：627.3[M+H] + 。
Step 3: s- (3- (((((3R, 5S) -1- ((R) -2- (3- (2, 2-diethoxyethoxy) isoxazol-5-yl) -3-methylbutanoyl) -5- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) carbonyl) oxy) -2-methylbutan-2-yl) methylsulfonate
Figure BDA0004113777320001662
To a catalyst containing (2S, 4R) -1- ((R) -2- (3- (2, 2-diethoxyethoxy) isoxazol-5-yl) -3-methylbutanoyl) -4-hydroxy-N- ((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) pyrrolidine-2-carboxamide (380 mg,0.62 mmol) and triethylamine (250 mg,2.47 mmol) and at 20℃over 16 hours
Figure BDA0004113777320001663
To a mixture of MS (50 mg) in anhydrous dichloromethane (5.0 mL) was slowly added S- (3- ((chlorocarbonyl) oxy) -2-methylbutan-2-yl) methylthiosulfonate (322 mg,1.24 mmol). The mixture was purified by preparative TLC (7% methanol in dichloromethane) to give the title compound (150 mg, 28.9%) as a white solid.
LCMS(ESI)m/z：839.7[M+H] + 。
Step 4: s- (2-methyl-3- (((((3R, 5S) -1- ((R) -3-methyl-2- (3- (2-oxoethoxy) isoxazol-5-yl) butanoyl) -5- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) carbonyl) butan-2-yl) methylsulfonate
Figure BDA0004113777320001671
A solution containing S- (3- (((((3R, 5S) -1- ((R) -2- (3- (2, 2-diethoxyethoxy) isoxazol-5-yl) -3-methylbutanoyl) -5- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) carbonyl) oxy) -2-methylbutan-2-yl) methylthiosulfonate (150 mg,0.18 mmol) in water (3.0 mL) and formic acid (8.0 mL) was stirred at 50℃for 2 hours. The reaction mixture was concentrated to dryness to give the title compound (135 mg, yield 98.7%) as a white solid.
LCMS(ESI)m/z：765.5[M+H] + 。
Step 5: s- (3- (((((3R, 5S) -1- ((2R) -2- (3- (2- ((3R) -4- (2- ((4- (3-amino-6- (2- ((phosphonooxy) methoxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazin-1-yl) ethoxy) isoxazol-5-yl) -3-methylbutanoyl) -5- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) -2-methylbutan-2-yl) methylthiosulfonate
Figure BDA0004113777320001681
To a solution of (2- (6-amino-5- (8- (2- (2- ((R) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) methyl dihydrogen phosphate (138 mg,0.19 mmol) and S- (2-methyl-3- (((((((3R, 5S) -1- ((R) -3-methyl-2- (3- (2-oxoethoxy) isoxazol-5-yl) butanoyl) -5- (((SS) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) butan-2-yl) methylthiosulfonate (130 mg,0.17 mmol) dichloromethane (5.0 mL) and methanol (5.0 mL) was added sodium triacetoxyborohydride (720 mg,3.40 mmol). The reaction mixture was stirred at 40 ℃ for 48 hours. The reaction mixture was purified by Column Welch Xtimate C, 150 x 25mm x 5um (over water (0.225% formic acid) -acetonitrile 20-50%) to give the title compound (54.2 mg, 21.3%) as a white solid.
LCMS(ESI)m/z：1375.5[M+H] + 。
1 H NMR(400MHz,DMSO-d 6 )δ(ppm)8.98(s,1H),8.52(d,J＝8.0Hz,1H),8.14(s,2H),7.74(d,J＝6.0Hz,1H),7.61(d,J＝8.0Hz,1H),7.47-7.30(m,5H),7.29-7.24(m,1H),7.03(t,J＝7.2Hz,1H),6.50(d,J＝6.0Hz,1H),6.26(s,1H),6.12(d,J＝2.4Hz,1H),5.79 -5.75(m,1H),5.51-5.46(m,2H),5.23-5.18(m,1H),5.06-4.85(m,3H),4.48-4.35(m,5H),4.26-4.23(m,2H),3.89-3.83(m,2H),3.75-3.71(m,2H),3.09-2.99(m,5H),2.97-2.87(m,4H),2.84-2.77(m,3H),2.73-2.71(m,2H),2.46-2.44(m,3H),2.30-2.11(m,5H),2.05-1.94(m,3H),1.57-1.35(m,9H),1.33-1.22(m,6H),1.11-1.04(m,4H),0.98-0.91(m,3H),0.86-0.77(m,3H)。
Synthesis example 10
Synthesis of L1-CIDE-BRM1-10
Figure BDA0004113777320001691
Step 1: s- (3- (((((3R, 5S) -1- ((2R) -2- (3- (2- ((3R) -4- (2- ((4- (3-amino-6- (2- ((phosphonooxy) methoxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazin-1-yl) ethoxy) isoxazol-5-yl) -3-methylbutanoyl) -5- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) butan-2-yl) methylthiosulfonate
Figure BDA0004113777320001692
To a solution of S- (3- ((((3R, 5S) -1- ((R) -3-methyl-2- (3- (2-oxoethoxy) isoxazol-5-yl) butanoyl) -5- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) pyrrolidin-3-yl) oxy) carbonyl) oxy) butan-2-yl) methylthiosulfonate (100 mg,0.13 mmol) and (2- (6-amino-5- (8- (2- (2- ((R) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) phenoxy) methylphosphonic acid dihydro salt (99.2 mg,0.16 mmol) dichloromethane (1.00 mL) and methanol (1.00 mL) was added sodium triacetoxyborohydride (615 mg,2.9 mmol). The reaction mixture was stirred at 20℃for 3 hours. The mixture was purified by Column Phenomenex Gemini-NX C18 75 x 30mm x 3um (10% -40% over water (0.225% formic acid) -acetonitrile) to give the title compound (110 mg, 51.4%) as a white solid.
LCMS(ESI)m/z：1375.5[M+H] + 。
1 H NMR(400MHz,DMSO-d 6 )δ(ppm)8.99(s,1H),8.51(d,J＝6.4Hz,1H),8.15(s,2H),7.82-7.72(m,1H),7.64(d,J＝7.6Hz,1H),7.52-7.32(m,6H),7.30-7.24(m,1H),7.08-7.03(m,1H),6.56-6.46(m,1H),6.33-6.30(m,1H),6.14(s,1H),5.91-5.86(m,1H),5.55-5.50(m,2H),5.21-5.18(m,1H),4.98-4.87(m,2H),4.51-4.31(m,5H),4.30-4.22(m,2H),3.91-3.84(m,2H),3.77-3.73(m,2H),3.58-3.51(m,2H),3.18-3.11(m,4H),3.07-2.97(m,4H),2.95-2.81(m,5H),2.79-2.72(m,2H),2.47-2.45(m,3H),2.37-2.14(m,5H),2.10-1.88(m,3H),1.48-1.23(m,9H),1.20-1.05(m,3H),0.98-0.93(m,3H),0.88-0.76(m,3H)。
Synthesis example 11
Synthesis of L1-CIDE-BRM1-11
Figure BDA0004113777320001701
Step 1: s- (3- ((chlorocarbonyl) oxy) butan-2-yl) methylthiosulfonate
Figure BDA0004113777320001702
To a solution of S- (3-hydroxybutyrin-2-yl) methylthiosulfonate (200 mg,1.09 mmol) and pyridine (343 mg,4.34 mmol) in methylene chloride (4.0 mL) was added methylene chloride (2.0 mL) containing triphosgene (129 mg,0.43 mmol). The reaction mixture was stirred at 25 ℃ for 30 minutes. The reaction mixture was concentrated to dryness to give the title compound (250 mg, yield 93.4%) as a yellow oil, which was used directly in the next step.
Step 2: s- (3- (((((3R, 5S) -5- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -1- ((R) -2- (3- (2, 2-diethoxyethoxy) isoxazol-5-yl) -3-methylbutanoyl) pyrrolidin-3-yl) oxy) carbonyl) oxy) butan-2-yl) methylthiosulfonate
Figure BDA0004113777320001711
To a mixture containing (2S, 4R) -N- ((S) -1- (4-cyanophenyl) ethyl) -1- ((R) -2- (3- (2, 2-diethoxyethoxy) isoxazol-5-yl) -3-methylbutyryl) -4-hydroxypyrrolidine-2-carboxamide (260 mg,0.48 mmol), triethylamine (194 mg,1.92 mmol) and
Figure BDA0004113777320001713
s- (3- ((chlorocarbonyl) oxy) butan-2-yl) methylthio sulfonic acid was slowly added to a mixture of MS (80 mg) in anhydrous DCM (4.0 mL)Acid salt (250 mg,1.01 mmol) of anhydrous dichloromethane (2.0 mL). The reaction mixture was filtered and the filtrate was purified by flash chromatography (silica gel, 100-200 mesh, petroleum ether containing 0-70% ethyl acetate) to give the title compound (150 mg, 41.6%) as a yellow oil.
LCMS(ESI)m/z：752.9[M+H] + 。
Step 3: s- (3- (((((3R, 5S) -5- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -1- ((R) -3-methyl-2- (3- (2-oxoethoxy) isoxazol-5-yl) butanoyl) pyrrolidin-3-yl) oxy) carbonyl) oxy) butan-2-yl) methylsulfonate
Figure BDA0004113777320001712
A solution containing S- (3- (((((3R, 5S) -5- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -1- ((R) -2- (3- (2, 2-diethoxyethoxy) isoxazol-5-yl) -3-methylbutanoyl) pyrrolidin-3-yl) oxy) carbonyl) oxy) butan-2-yl) methylthiosulfonate (150 mg,0.20 mmol) of formic acid (5.0 mL) and water (1.0 mL) was stirred at 50℃for 16 hours. The reaction mixture was concentrated to dryness to give the title compound (100 mg, yield 73.9%) as a yellow oil.
LCMS(ESI)m/z：679.5[M+H] + 。
Step 4: s- (3- (((((3R, 5S) -1- ((2R) -2- (3- (2- (4- ((1R, 3R) -3- ((4- (3-amino-6- (2- ((phosphonooxy) methoxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) cyclobutoxy) piperidin-1-yl) ethoxy) isoxazol-5-yl) -3-methylbutyl) -5- (((S) -1- (4-cyanophenyl) ethyl) pyrrolidin-3-yl) oxy) butan-2-yl) methylthiosulfonate
Figure BDA0004113777320001721
To a solution of methyl (2- (6-amino-5- (8- (2- ((1R, 3R) -3- (piperidin-4-yloxy) cyclobutoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) phosphate (169 mg,0.22 mmol) and sodium triacetoxyborohydride (1.40 g,6.63 mmol) in methylene chloride (5.0 mL) and methanol (5.0 mL) were added. The reaction mixture was stirred at 20 ℃ for 48 hours. The reaction mixture was purified by Column Phenomenex Gemini-NX C18 75 x 30mm x 3um (over water (0.225% formic acid) -acetonitrile 10-40%) to give the title compound (25.6 mg, 8.8% yield) as a white solid.
LCMS(ESI)m/z：659.1[M/2+H] + 。
1 H NMR(400MHz,DMSO-d 6 )δ(ppm)8.58(d,J＝8.0Hz,1H),8.14(s,1H),7.82-7.74(m,3H),7.55(d,J＝8.0Hz,1H),7.50-7.43(m,3H),7.39-7.34(m,1H),7.22(s,1H),7.15-7.09(m,1H),6.54-6.51(m,1H),6.37-6.21(m,2H),6.16-6.10(m,2H),5.57-5.52(m,2H),5.18-5.12(m,2H),4.99-4.87(m,2H),4.52-4.44(m,2H),4.40-4.22(m,4H),3.87-3.82(m,2H),3.74-3.70(m,1H),3.60-3.50(m,2H),3.15-3.00(m,2H),2.96-2.79(m,4H),2.69-2.64(m,1H),2.35-2.18(m,9H),2.16-2.09(m,2H),2.02-1.74(m,6H),1.53-1.23(m,12H),0.95-0.91(m,3H),0.85-0.74(m,3H)。
Synthesis example 12
Synthesis of L1-CIDE-BRM1-12
Figure BDA0004113777320001731
Step 1: s- (3- (((((3R, 5S) -5- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -1- ((R) -2- (3- (2, 2-diethoxyethoxy) isoxazol-5-yl) -3-methylbutanoyl) pyrrolidin-3-yl) oxy) carbonyl) oxy) -2-methylbutan-2-yl) methylsulfonate
Figure BDA0004113777320001732
To a mixture containing (2S, 4R) -N- ((S) -1- (4-cyanophenyl) ethyl) -1- ((R) -2- (3-)(2, 2-Diethoxyethoxy) isoxazol-5-yl) -3-methylbutyryl) -4-hydroxypyrrolidine-2-carboxamide (300 mg,0.55 mmol), pyridine (175 mg,2.21 mmol) and
Figure BDA0004113777320001733
to a mixture of dry dichloromethane (5.0 mL) of MS (100 mg) was slowly added dry dichloromethane (2 mL) containing S- (3- ((chlorocarbonyl) oxy) -2-methylbutan-2-yl) methylthiosulfonate (319 mg,1.00 mmol). The reaction mixture was purified by preparative TLC (7% methanol in dichloromethane) to give the title compound (60.0 mg, 14.2%) as a white solid.
LCMS(ESI)m/z：722.1[M+H] + 。
Step 3: s- (3- (((((3R, 5S) -5- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -1- ((R) -3-methyl-2- (3- (2-oxoethoxy) isoxazol-5-yl) butanoyl) pyrrolidin-3-yl) oxy) carbonyl) oxy) 2-methylbutan-2-yl) methylsulfonate
Figure BDA0004113777320001741
A solution containing S- (3- (((((3R, 5S) -5- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -1- ((R) -2- (3- (2, 2-diethoxyethoxy) isoxazol-5-yl) -3-methylbutanoyl) oxy) pyrrolidin-3-yl) oxy) carbonyl) oxy) -2-methylbutan-2-yl) methylthiosulfonate (60.0 mg,0.08 mmol) in water (1.00 mL) and formic acid (5.00 mL). The reaction mixture was stirred at 50℃for 2 hours. The resulting residue was concentrated to give the title compound (50 mg, 92.2%) as a white solid. LCMS (ESI) m/z:693.2[ M+H ] ] + 。
Step 4: s- (3- (((((3R, 5S) -1- ((2R) -2- (3- (2- (4- ((1R, 3R) -3- ((4- (3-amino-6- (2- ((phosphonooxy) methoxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) cyclobutoxy) piperidin-1-yl) ethoxy) isoxazol-5-yl) -3-methylbutanoyl) -5- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) oxo) -2-methylbutan-2-yl) methylthiosulfonate
Figure BDA0004113777320001742
To a solution of (2- (6-amino-5- (8- (2- ((1R, 3R) -3- (piperidin-4-yloxy) cyclobutoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) phosphate (138.51 mg,0.18 mmol) and S- (3- (((((3R, 5S) -5- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -1- ((R) -3-methyl-2- (3- (2-oxoethoxy) isoxazol-5-yl) butyryl) pyrrolidin-3-yl) oxy) carbonyl) oxy) -2-methylbutan-2-yl) methylthiosulfonate (125 mg,0.18 mmol) in dichloromethane (5.00 mL) and methanol (5.00 mL) was added sodium triacetoxyborohydride (38.0.0.18 mmol). The reaction mixture was stirred at 20℃for 36 hours. The reaction mixture was purified by Column Welch Xtimate C, 150 x 25mm x 5um (over water (0.225% formic acid) -acetonitrile 17-47%) to give the title compound (15.9 mg, 50.4% yield) as a white solid.
LCMS(ESI)m/z：666.1[M/2+H] + 。
1 H NMR(400MHz,DMSO-d 6 )δ(ppm)8.61-8.56(m,1H),7.81-7.73(m,3H),7.57-7.42(m,4H),7.38-7.32(m,1H),7.22(s,1H),7.15-7.08(m,1H),6.54-6.50(m,1H),6.29-6.25(m,2H),6.15-6.10(m,2H),5.58-5.52(m,2H),5.22-5.11(m,2H),5.04-5.01(m,1H),4.94-4.91(m,1H),4.49-4.45(m,2H),4.41-4.36(m,1H),4.28-4.25(m,3H),3.90-3.82(m,2H),3.75-3.71(m,2H),3.56-3.51(m,1H),3.10-2.82(m,5H),2.31-2.18(m,7H),2.16-2.09(m,2H),2.02-1.77(m,5H),1.56-1.40(m,11H),1.39-1.20(m,8H),1.16-1.12(m,1H),0.95-0.91(m,3H),0.85-0.76(m,4H)。
Synthesis example 13
Synthesis of L1-CIDE-BRM1-13
Figure BDA0004113777320001751
Step 1: (S) -1- ((1- ((4- (chloromethyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylic acid ethyl ester
Figure BDA0004113777320001761
To a solution of ethyl (S) -1- ((1- ((4- (hydroxymethyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylate (1.4 g,3.22 mmol) in dichloromethane (50.0 mL) and NMP (1.0 mL) was added thionyl chloride (0.70 mL,9.67 mmol) at 25 ℃. The reaction mixture was stirred at 25 ℃ for 3 hours. The reaction mixture was purified by flash chromatography (silica gel, 100-200 mesh, dichloromethane solution containing 0-5% methanol) to give the title compound (1.40 g, 96% yield) as a yellow oil.
Step 2: (S) -1- ((1- ((4- ((2-bromophenoxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylic acid ethyl ester
Figure BDA0004113777320001762
To a mixture containing (S) -1- ((1- ((4- (chloromethyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) ethyl cyclobutanecarboxylate (1.40 g,3.09 mmol) and potassium carbonate at 25 ℃ 3 To a solution of (1.07 g,7.73 mmol) in N, N-dimethylformamide (60 mL) was added 2-bromophenol (0.54 mL,4.64 mmol). The reaction was stirred at 25℃for 3 hours. The reaction was diluted with water (30.0 mL) and extracted with dichloromethane (50.0 mL x 3). The combined organic layers were washed with brine (20.0 ml x 2), dried over sodium sulfate, filtered, and concentrated to dryness. The residue was purified by flash chromatography (silica gel, 100-200 mesh, dichloromethane containing 0-5% methanol) to give the title compound (1.1 g, yield 60%) as a white solid.
LCMS(ESI)m/z：590.7[M+H] + 。
Step 3: (S) -1- ((1-oxo-1- ((4- ((2- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenoxy) methyl) phenyl) amino) -5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylic acid ethyl ester
Figure BDA0004113777320001771
To a solution of ethyl (S) -1- ((1- ((4- ((2-bromophenoxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylate (1.10 g,1.87 mmol) and bis (pinacolato) diboron (711 mg,2.80 mmol) in dimethyl sulfoxide (20.0 mL) was added Pd (dppf) Cl 2 (137 mg,0.19 mmol) and potassium acetate (549 mg,5.60 mmol). The mixture is put under N 2 Stirring at 100℃for 3 hours. The crude mixture was used directly in the next step.
LCMS(ESI)m/z：637.1[M+H] + 。
Step 4: (3R) -4- (2- ((4- (3-amino-6- (2- ((4- ((S) -2- (1- (ethoxycarbonyl) cyclobutanecarboxamido) -5-ureidovaleramido) benzyl) oxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester
Figure BDA0004113777320001772
To (3R) -4- (2- ((4- (3- (3-amino-6-chloropyridazin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]Octane-8-yl) pyridin-2-yloxy) ethyl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester (750 mg,1.28 mmol) and tripotassium orthophosphate (814 mg,3.84 mmol) dimethyl sulfoxide (15.0 mL) and H 2 To a solution of O (1.00 mL) was added chloro [ (di (1-adamantyl) -n-butylphosphine) -2- (2-aminobiphenyl)]Palladium (II) (171 mg,0.26 mmol) and ethyl (S) -1- ((1-oxo-1- ((4- ((2- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenoxy) methyl) phenyl) amino) -5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylate (830 mg,1.31 mmol). The mixture is put under N 2 Stirring at 100℃for 3 hours. The reaction was purified by silica gel column chromatography (silica gel, 100-200 mesh, dichloromethane solution containing 0-15% methanol) to give the title compound (400 mg, yield 28%) as a black oil.
LCMS(ESI)m/z：1251.0[M+H] + 。
Step 5:1- (((2S) -1- ((4- ((2- (6-amino-5- (8- (2- (2- ((R) -4- (tert-butoxycarbonyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylic acid
Figure BDA0004113777320001781
To a compound containing (3R) -4- (2- ((4- (3- (3-amino-6- (2- ((4- ((S) -2- (1- (ethoxycarbonyl) cyclobutanecarboxamido) -5-ureidovaleramido) benzyl) oxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]To a solution of tert-butyl octan-8-yl) pyridin-2-yloxy) -3-methylpiperazine-1-carboxylate (400 mg,0.39 mmol) in methanol (5.0 mL) and water (2.0 mL) was added lithium hydroxide monohydrate (92.7 mg,3.87 mmol). The mixture is put under N 2 Stirred at 25℃for 3 hours. The reaction mixture was concentrated to dryness to give the title compound (300 mg, yield 77.1%) as a black solid. LCMS (ESI) m/z:1005.6[ M+H ]] + 。
Step 6:1- (((2S) -1- ((2, 2-trifluoro-acetic acid 2, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylic acid 2, 2-amino-5- (8- (2- (2- ((R) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) phenoxy) methyl) phenyl
Figure BDA0004113777320001782
To a solution of 1- (((2S) -1- ((4- ((2- (6-amino-5- (8- (2- (2- ((R) -4- (tert-butoxycarbonyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) phenoxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylic acid (300 mg,0.3 mmol) in dichloromethane (20.0 mL) was added trifluoroacetic acid (0.20 mL,3.00 mmol). The solution was stirred at 20 ℃ for 5 hours and then concentrated to dryness. The crude product was purified by preparative HPLC using the following conditions: (column: welch Xtimate C18 100X 40mm X3 um; mobile phase: 12-42% water (0.075% trifluoroacetic acid-acetonitrile) to give the title compound (89 mg, 32.9%) as a white solid.
LCMS(ESI)m/z：905.5[M+H] + 。
Step 7:1- (((2S) -1- ((2- (6-amino-5- (8- (2- (2- ((R) -4- (2- ((5- ((R) -1- ((2S, 4R) -4-hydroxy-2- (((S) -1- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) isoxazol-3-yl) oxy) ethyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylic acid
Figure BDA0004113777320001791
To a solution containing (2S, 4R) -4-hydroxy-1- ((R) -3-methyl-2- (3- (2-oxoethoxy) isoxazol-5-yl) butanoyl) -N- ((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) pyrrolidine-2-carboxamide (86.02 mg,0.16 mmol) and 1- (((2S) -1- ((4- ((2- (6-amino-5- (8- (2- ((R) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]Octane-3-yl) pyridazin-3-yl) phenoxy methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl carbamoyl) cyclobutanecarboxylic acid 2, 2-trifluoroacetate (96.0 mg,0.11 mmol) in dichloromethane (1.50 mL) and methanol (1.50 mL) was added sodium cyanoborohydride (13.6 mg,0.21 mmol). The reaction mixture was stirred at 20℃for 3 hours. The resulting solution was passed through Phenomenex Gemini-NX 80 x 40mm x 3um (acetonitrile 17-47%/0.05% NH) 3 H 2 O aqueous solution) to give the title compound (89.0 mg, yield 58.7%) as a white solid. LCMS (ESI) m/z:1429.9[ M+H ]] + 。
Step 8: n- ((2S) -1- ((4- ((2- (6-amino-5- (8- (2- (2- ((R) -4- (2- ((5- ((R) -1- ((2S, 4R) -4-hydroxy-2- (((S) -1- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) oxy) isoxazol-3-yl) oxy) ethyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) -N- (5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) cyclobutane-1, 1-dicarboxamide
Figure BDA0004113777320001801
To a catalyst comprising 1- (((2S) -1- ((2- (6-amino-5- (8- (2- (2- ((R) -4- (2- ((R) -1- ((2S, 4R) -4-hydroxy-2- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) isoxazol-3-yl) oxy) ethyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1]To a mixture of octane-3-yl-pyridazin-3-yl-phenoxy) methyl) phenyl) -amino-1-oxo-5-ureidopentan-2-yl-carbamoyl) cyclobutanecarboxylic acid (50.0 mg,0.03 mmol) and 1- (5-aminopentyl) -1H-pyrrole-2, 5-dione 2, 2-trifluoro-acetic acid (12.4 mg,0.04 mmol) in N, N-dimethylformamide (4.0 mL) was added N, N-diisopropylethylamine (0.02 mL,0.10 mmol) and 2- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate (16.0 mg,0.04 mmol). The mixture was stirred at 25℃for 3 hours. The reaction mixture was concentrated to dryness by an oil pump. The residue was purified by preparative HPLC (Boston Green ODS 150 x 30mm x 5um, water (0.075% trifluoroacetic acid) -acetonitrile, 20-50%) to give the title compound (33.6 mg, 59.7%) as a white solid. 1 H NMR(400MHz,DMSO-d 6 ):δ(ppm)10.19(s,1H),9.02-8.86(m,1H),8.39(d,J＝8.0Hz,1H),7.94(d,J＝6.8Hz,1H),7.87-7.78(m,2H),7.65(d,J＝8.0Hz,2H),7.51-7.41(m,5H),7.36(d,J＝6.8Hz,5H),7.29-7.09(m,1H),6.97(s,2H),6.76(s,1H),6.42(s,1H),6.15-5.96(m,2H),5.07(s,2H),4.93-4.86(m,1H),4.71-4.60(m,2H),4.52-4.24(m,9H),3.66(s,4H),3.33(d,J＝7.2Hz,5H),3.09-2.96(m,8H),2.45(s,3H),2.42-2.34(m,4H),2.29-2.14(m,2H),2.04(d,J＝11.2Hz,3H),1.91(s,2H),1.83-1.55(m,5H),1.51-1.30(m,9H),1.19(d,J＝5.8Hz,5H),0.96(d,J＝6.4Hz,3H),0.86-0.75(m,3H)。
LCMS(ESI)m/z：797.5[M/2+H] + 。
Synthesis example 14
Synthesis of L1-CIDE-BRM1-14
Figure BDA0004113777320001811
Step 1:4- ((1 r,3 r) -3- ((4- (3-amino-6- (2- ((4- ((S) -2- (1- (ethoxycarbonyl) cyclobutanecarboxamido) -5-ureidovaleramido) benzyl) oxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) cyclobutoxy) piperidine-1-carboxylic acid tert-butyl ester
Figure BDA0004113777320001812
To a compound containing 4- ((1 r,3 r) -3- ((4- (3- (3-amino-6-chloropyridazin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]To a solution of tert-butyl octan-8-yl-pyridin-2-yloxy) cyclobutoxy-piperidine-1-carboxylate (450 mg,0.77 mmol) and tripotassium orthophosphate (0.19 mL,2.3 mmol) in dimethyl sulfoxide (20 mL) was added chloro [ (di (1-adamantyl) -n-butylphosphine) -2- (2-aminobiphenyl)]Palladium (II) (103 mg,0.15 mmol) and ethyl (S) -1- ((1-oxo-1- ((4- ((2- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenoxy) methyl) phenyl) amino) -5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylate (977 mg,1.54 mmol). The mixture is put under N 2 Stirring at 100℃for 3 hours. The reaction mixture was diluted with water (10 mL) and extracted with dichloromethane (20 mL x 3). The organic phase was washed with brine (30 ml x 2), dried over sodium sulfate, filtered and concentrated to give the title compound (400 mg, 49.1%) as a black solid.
LCMS(ESI)m/z：1060.7[M+H] + 。
Step 2:1- (((2S) -1- ((4- ((2- (6-amino-5- (8- (2- ((1 r,3 r) -3- ((1- (tert-butoxycarbonyl) piperidin-4-yl) oxy) cyclobutoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylic acid
Figure BDA0004113777320001821
To a composition containing 4- ((1 r,3 r) -3- ((4- (3-amino-6- (2- ((4- ((S))2- (1- (ethoxycarbonyl) cyclobutanecarboxamido) -5-ureidovaleramido) benzyl) oxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1]To a solution of butyl octan-8-yl pyridin-2-yl) oxy-cyclobutoxy-piperidine-1-carboxylate (450 mg,0.42 mmol) in methanol (5.0 mL) and water (2.0 mL) was added lithium hydroxide monohydrate (102 mg,4.24 mmol). The mixture was stirred at 25℃for 3 hours. The reaction mixture was concentrated to give the title compound (438 mg, yield 97.5%) as a black solid. LCMS (ESI) m/z:1032.6[ M+H ]] + 。
Step 3:1- (((2S) -1- ((2, 2-trifluoroacetate) amino) -1-oxo-5- (8- (2- ((1 r,3 r) -3- (piperidin-4-yloxy) cyclobutoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylic acid 2,2
Figure BDA0004113777320001822
To a mixture of 1- (((2S) -1- ((4- ((2- (6-amino-5- (8- (2- ((1 r,3 r) -3- ((1- (tert-butoxycarbonyl) piperidin-4-yl) oxy) cyclobutoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylic acid (400 mg,0.39 mmol) in dichloromethane (4.0 mL) was added trifluoroacetic acid (0.30 mL,3.90 mmol). The mixture was stirred at 20℃for 5 hours. The reaction was concentrated to dryness and the residue was purified by preparative HPLC (Boston Green ODS 150 x 30mm x 5um, water (0.075% trifluoroacetic acid) -acetonitrile 12% -42%) to give the title compound (360 mg, 88.9% yield).
LCMS(ESI)m/z：932.6[M+H] + 。
Step 4:1- (((2S) -1- ((4- ((2- (6-amino-5- (8- (2- ((1R, 3R) -3- ((1- (2- ((5- ((R) -1- ((2S, 4R) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -4-hydroxypyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) oxy) isoxazol-3-yl) oxy) ethyl) piperidin-4-yl) oxy) cyclobutoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) methyl) phenyl) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylic acid
Figure BDA0004113777320001831
To a solution containing (2S, 4R) -N- ((S) -1- (4-cyanophenyl) ethyl) -4-hydroxy-1- ((R) -3-methyl-2- (3- (2-oxoethoxy) isoxazol-5-yl) butanoyl) pyrrolidine-2-carboxamide (313.21 mg,0.5800 mmol) and 1- (((2S) -1- ((2- (6-amino-5- (8- (2- ((1R, 3R) -3- (piperidin-4-yloxy) cyclobutoxy) pyridin-4-yl) -3, 8-diazabicyclo [ 3.2.1) ]Octane-3-yl) pyridazin-3-yl) phenoxy methyl) phenyl) -amino) -1-oxo-5-ureidopentan-2-yl carbamoyl) cyclobutanecarboxylic acid 2, 2-trifluoroacetate (360 mg,0.39 mmol) in dichloromethane (0.50 mL) and methanol (0.50 mL) was added sodium cyanoborohydride (49.3 mg,0.77 mmol) and sodium acetate (6.00 mg,0.07 mmol) as one drop of acetic acid. The reaction mixture was stirred at 20℃for 3 hours. Purification of the residue using Phenomenex Gemini-NX 80X 40mm X3 um (acetonitrile 17-47/0.05% NH) 3 H 2 O aqueous solution, 20% -50%) to give the title compound as a white solid (250 mg, 46.7%).
LCMS(ESI)m/z：1384.8[M+H] + 。
Step 5: n- ((2S) -1- ((4- ((2- (6-amino-5- (8- (2- ((1R, 3R) -3- ((1- (2- ((5- ((R) -1- ((2S, 4R) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -4-hydroxypyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) oxy) isoxazol-3-yl) oxy) ethyl) piperidin-4-yl) oxy) cyclobutoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) -N- (5- (2, 5-dihydro-1H-pyrrol-1-yl) pentyl) cyclobutane-1, 1-dicarboxamide
Figure BDA0004113777320001832
Figure BDA0004113777320001841
To a mixture of N- (4.02 mL) of N- (2.02 mL) benzotriazol-7-yl) 2, 2mL containing 1- (((2S) -1- ((2- (6-amino-5- (8- ((2- ((1R, 3R) -3- ((1- ((R) -1- ((2S, 4R) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -4-hydroxypyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) oxy) isoxazol-3-yloxy) ethyl) piperidin-4-yloxy) cyclobutoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) phenoxy) methyl) amino) -1-oxo-5-ureido-pentan-2-yl carbamoyl) cyclobutanecarboxylic acid (50.0 mg,0.04 mmol) and 1- (5-aminopentyl) -1H-pyrrole-2, 5-dione 2, 2-trifluoroacetate (12.8 mg,0.04 mmol) N-dimethyl-N- (4.02 mL) benzotriazol-3-yl) N- (0.02 mL, n, N ', N' -tetramethyl urea hexafluorophosphate (16.5 mg,0.04 mmol). The mixture was stirred at 25℃for 3 hours. The mixture was concentrated by an oil pump. The residue was purified by preparative HPLC (Boston Green ODS 150 x 30mm x 5um, water (0.075% trifluoroacetic acid) -acetonitrile 20% -50%) to give the title compound (38.5 mg, 65.4%) as a white solid.
LCMS(ESI)m/z：775.3[M/2+H] + 。
1 H NMR(400MHz,DMSO-d 6 )δ(ppm)10.19(s,1H),9.02-8.86(m,1H),8.39(d,J＝8.0Hz,1H),7.94(d,J＝6.8Hz,1H),7.87-7.78(m,2H),7.65(d,J＝8.0Hz,2H),7.51-7.41(m,5H),7.36(d,J＝6.8Hz,5H),7.29-7.09(m,1H),6.97(s,2H),6.76(s,1H),6.42(s,1H),6.15-5.96(m,2H),5.07(s,2H),4.93-4.86(m,1H),4.71-4.60(m,2H),4.52-4.24(m,9H),3.66(s,4H),3.33(d,J＝7.2Hz,5H),3.09-2.96(m,8H),2.45(s,3H),2.42-2.34(m,4H),2.29-2.14(m,2H),2.04(d,J＝11.2Hz,3H),1.91(s,2H),1.83-1.55(m,5H),1.51-1.30(m,9H),1.19(d,J＝5.8Hz,5H),0.96(d,J＝6.4Hz,3H),0.86-0.75(m,3H)。
Synthesis example 15
Synthesis of L1-CIDE-BRM1-15
Figure BDA0004113777320001851
Step 1: (3R) -4- (2- ((4- (3- (3-amino-6- (2- ((fluorosulfonyl) oxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester
Figure BDA0004113777320001852
At SO 2 F 2 Under the balloon, the composition contains (3R) -4- (2- ((4- (3- (3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]A solution of tert-butyl octan-8-yl) pyridin-2-yloxy) -3-methylpiperazine-1-carboxylate (500 mg,0.81 mmol) in dichloromethane (3.0 mL) was stirred at 0deg.C for 16 h. The mixture was concentrated to give the title compound (566 mg, 99.8%) as a yellow oil. The crude product was used directly in the next step. LCMS (ESI) m/z:713.5[ M+Na] + 。
Step 2: n- (2- (2- (2-azidoethoxy) ethoxy) ethyl) -3- ((tert-butyldimethylsilyl) oxy) -4- (((2S, 3R,4S,5R, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) benzamide
Figure BDA0004113777320001853
To a solution of N- (2- (2- (2- (2-azidoethoxy) ethoxy) ethyl) -3-hydroxy-4- (((2S, 3R,4S,5R, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) benzamide (320 mg,0.70 mmol) in dichloromethane (10.0 mL) was added 2, 6-dimethylpyridine (0.24 mL,2.09 mmol) and tert-butyldimethylsilyl triflate (0.32 mL,1.40 mmol) and stirred at 0deg.C for 2 hours. The reaction was concentrated and the residue was purified using Column Phenomenex Gemini-NX C18 75 x 30mm x 3um (purified over water (0.05% NH 3 H 2 O+10mM NH 4 HCO 3 ) Acetonitrile (35% -65%)) to give the title compound (140 mg, 35%) as a white solid.
LCMS(ESI)m/z：573.4[M+H] + 。
Step 3: (3R) -4- (2- ((4- (3- (3-amino-6- (2- (((5- ((2- (2- (2- (2-azidoethoxy) ethoxy) ethyl) carbamoyl) -2- (((2S, 3R,4S,5R, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) phenoxy) sulfonyl) oxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester
Figure BDA0004113777320001861
Acetonitrile (0.86 mL,0.86 mmol) solution containing 1M 2-tert-butylimino-2-diethylamino-1, 3-dimethyl-perhydro-1, 3, 2-diazaphosphine, (3R) -4- (2- ((4- (3-amino-6- (2- ((fluorosulfonyl) oxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]Octane-8-yl) pyridin-2-yloxy) ethyl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester (300 mg,0.43 mmol) and a solution of N- (2- (2- (2- (2-azidoethoxy) ethoxy) ethyl) -3- ((tert-butyldimethylsilyl) oxy) -4- (((2S, 3R,4S,5R, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) benzamide (274 mg,0.43 mmol) in acetonitrile (5.0 mL) and N, N-dimethylformamide (1.00 mL) were stirred at 24℃for 16 hours. The crude mixture was concentrated to dryness and the residue was purified by prep HPLC (Welch Xtimate C18 x 25mM x 5 um/water (10 mM NH 4 HCO 3 ) Acetonitrile/40-70%) to give the title compound as a white solid (200 mg, 39% yield).
LCMS(ESI)m/z：1195.6[M+H] + 。
Step 4:2- (6-amino-5- (8- (2- (2- ((R) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenyl (5- ((2- (2- (2-azidoethoxy) ethoxy) ethyl) carbamoyl) -2- (((2S, 3R,4S,5R, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) phenyl) sulfate 2, 2-trifluoroacetate
Figure BDA0004113777320001871
A solution of tert-butyl (5% (205 mg,0.17 mmol) of 5% trifluoroacetic acid in hexafluoroisopropanol (6.00 mL) containing (3R) -4- (2- ((4- (3-amino-6- (2- (((2- (2-azidoethoxy) ethoxy) ethyl) carbamoyl) -2- (((2S, 3R,4S,5R, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) phenoxy) sulfonyl) oxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazine-1-carboxylate was stirred at 15℃for 2 hours. The mixture was concentrated to give the title compound (207 mg, 99.8%) as a white solid. The crude product was used directly in the next step.
LCMS(ESI)m/z：1095.4[M+H-TFA] + 。
Step 4:2- (6-amino-5- (8- (2- ((R) -4- (2- ((5- ((R) -1- ((2S, 4R) -4-hydroxy-2- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) isoxazol-3-yl) oxy) ethyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenyl (5- ((2- (2- (2- (2-azidoethoxy) ethoxy) ethyl) carbamoyl) -2- (((2S, 3R,4S,5R, 6R) -3, 4-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) phenyl) sulfate
Figure BDA0004113777320001872
To a compound containing 2- (6-amino-5- (8- (2- (2- ((R) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]Octane-3-yl) pyridazin-3-yl) phenyl (5- ((2- (2- (2- (2-azidoethoxy) ethoxy) ethyl) carbamoyl) -2- (((2S, 3R,4S,5R, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) phenyl) sulfate 2, 2-trifluoroacetate (207 mg,0.17 mmol) and (2S, 4R) -4-hydroxy-1- ((R) -3-methyl-2- (3- (2-oxoethoxy) isoxazol-5-yl) butyryl) -N- ((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) pyrrolidine-2-carboxamide (184 mg,0.34 mmol) in methanol (1.00 mL) and dichloromethane (1.00 mL) was added sodium cyanoborohydride (11.8 mg,0.19 mmol) and sodium acetoxy (42.1 mg,0.51 mmol). The reaction was stirred at 20℃for 3 hours. The crude product was purified by prep HPLC (Welch xlmate C18 x 25mM x 5 um/water (10 mM NH 4 HCO 3 ) Acetonitrile/30-60%) to give the title compound (145 mg, 52.3%) as a white solid. LCMS (ESI) m/z:810.9[1/2M+H]+。
Step 5:2- (6-amino-5- (8- (2- ((R) -4- (2- ((5- ((R) -1- ((2S, 4R) -4-hydroxy-2- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) isoxazol-3-yl) oxy) ethyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1 octan-3-yl) pyridazin-3-yl) phenyl (5- ((2- (2- (2- (4- (17- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -15-oxo-2, 5,8, 11-tetraoxa-14-azaheptadecyl) -1H-1,2, 3-triazol-1-yl) ethoxy) ethyl) carbamoyl) -2- (((2S, 3R,4S,5R, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H) -pyran-2-yl) oxy) phenyl) sulphate
Figure BDA0004113777320001881
2- (6-amino-5- (8- (2- ((R) -4- (2- ((5- ((R) -1- ((2S, 4R) -4-hydroxy-2- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) isoxazol-3-yl) oxy) ethyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1 ] at 0deg.C]Octane-3-yl) pyridazin-3-yl) phenyl (5- ((2- (2- (2- (2-azidoethoxy) ethoxy) ethyl) carbamoyl) -2- (((2S, 3R,4S,5R, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) phenyl) sulfate (45.0 mg,0.03 mmol) and 3- (2, 5-dioxopyrrol-1-yl) -N- [2- [2- [2- (2-prop-2-ynyloxyethoxy) ethoxy]Ethoxy group]Ethyl group]To a solution of propionamide (20.0 mg,0.05 mmol) in dimethyl sulfoxide (1.00 mL) and water (1.00 mL) was added a solution of copper sulfate (12.4 mg,0.06 mmol) in water (0.2 mL) and a solution of sodium ascorbate (11.01 mg,0.06 mmol) in water (0.2 mL). The reaction is carried out in N 2 Stirring is carried out for 1 hour at 26℃under an atmosphere. The crude product was purified by preparative HPLC (Welch xomate C18 x 40mm x 3 um/water (0.075% trifluoroacetic acid) -acetonitrile/15-45%) to give the title compound (22.6 mg, 40.6%) as a white solid.
LCMS(ESI)m/z：1001.9[1/2M+H] + 。
Synthesis example 16
Synthesis of L1-CIDE-BRM1-16
Figure BDA0004113777320001891
Step 1: (S) -1- ((1- ((4- ((2-bromo-4-fluorophenoxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylic acid ethyl ester
Figure BDA0004113777320001892
To a solution of ethyl (S) -1- ((1- ((4- (chloromethyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylate (1.00 g,2.21 mmol) and potassium carbonate (0.76 g,5.52 mmol) in N, N-dimethylformamide (60.0 mL) was added 2-bromo-4-fluoro-phenol (0.63 g,3.31 mmol) at 25 ℃. The reaction was stirred at 25℃for 3 hours. The reaction mixture was diluted with water (30.0 mL) and extracted with dichloromethane (50 mL x 3). The combined organic phases were washed with brine (20 ml x 2), dried over sodium sulfate, filtered, and concentrated to dryness. The residue was purified by flash chromatography (silica gel, 100-200 mesh, dichloromethane containing 0-5% methanol) to give the title compound (1.2 g, 89.5%) as a white solid.
Step 2: (S) -1- ((1- ((4- ((4-fluoro-2- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenoxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylic acid ethyl ester
Figure BDA0004113777320001901
To a mixture of ethyl (S) -1- ((1- ((4- ((2-bromo-4-fluorophenoxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylate (1.00 g,1.65 mmol) and 4,4', 5' -octamethyl-2, 2' -bis (1, 3, 2-dioxa) To a solution of cyclopentylborane) (627 mg,2.47 mmol) in 1, 4-dioxane (5.0 mL) was added 1,1' -bis (diphenylphosphino) ferrocene palladium dichloride (120 mg,0.16 mmol) and sodium acetate (481mg, 4.94 mmol). The mixture is put under N 2 Stirring at 100℃for 3 hours. The crude mixture was used directly in the next step.
LCMS(ESI)m/z：655.4[M+H] + 。
Step 3: (3R) -4- (2- ((4- (3-amino-6- (2- ((4- ((S) -2- (1- (ethoxycarbonyl) cyclobutanecarboxamido) -5-ureidovaleramido) benzyl) oxy) -5-fluorophenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester
Figure BDA0004113777320001902
To (3R) -4- (2- ((4- (3- (3-amino-6-chloropyridazin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]To a solution of tert-butyl octan-8-yl-pyridin-2-yloxy) -ethyl) -3-methylpiperazine-1-carboxylate (1.11 g,1.99 mmol) and potassium carbonate (0.38 mL,4.58 mmol) in dimethyl sulfoxide (5 mL) was added [2- (2-aminophenyl) phenyl ]]-chloro-palladium; bis (1-adamantyl) -butyl-phosphine (102.15 mg,0.15 mmol) and (S) -1- ((ethyl 1- ((4- ((4-fluoro-2- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenoxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylate (1.00 g,1.53 mmol). The reaction mixture was taken up in N 2 Stirring at 100℃for 3 hours. The reaction was purified by flash chromatography (silica gel, 100-200 mesh, 0-10% methanol in dichloromethane) to give the title compound (360 mg, 22.4%) as a black solid.
LCMS(ESI)m/z：1095[M+H] + 。
Step 4:1- (((2S) -1- ((4- ((2- (6-amino-5- (8- (2- (2- ((R) -4- (tert-butoxycarbonyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) -4-fluorophenoxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylic acid
Figure BDA0004113777320001911
To a compound containing (3R) -4- (2- ((4- (3- (3-amino-6- (2- ((4- ((S) -2- (1- (ethoxycarbonyl) cyclobutanecarboxamido) -5-ureidovaleramido) benzyl) oxy) -5-fluorophenyl) pyridazin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]To a solution of tert-butyl octan-8-yl) pyridin-2-yloxy) -3-methylpiperazine-1-carboxylate (360 mg,0.34 mmol) in tetrahydrofuran (2.00 mL), methanol (5.0 mL) and water (2.00 mL) was added lithium hydroxide monohydrate (41.0 mg,1.71 mmol). The reaction mixture was taken up in N 2 Stirring is carried out for 3 hours at 100℃under an atmosphere. The reaction mixture was concentrated to dryness to give the title compound (350 mg, 99.9%) as a white solid.
Step 5:1- (((2S) -1- ((2, 6-amino-5- (8- (2- (2- ((R) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) -4-fluorophenoxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylic acid 2, 2-trifluoro acetate
Figure BDA0004113777320001912
To a solution of 1- (((2S) -1- ((4- ((2- (6-amino-5- (8- (2- (2- ((R) -4- (tert-butoxycarbonyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) -4-fluorophenoxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylic acid (340 mg,0.33 mmol) in dichloromethane (5.0 mL) was added trifluoroacetic acid (0.05 mL,0.66 mmol). The reaction mixture was stirred at 20℃for 3 hours. The reaction mixture was concentrated to dryness and the residue was purified by preparative HPLC (Boston Green ODS 150 x 30mm x 5um, water (0.075% trifluoroacetic acid) -acetonitrile 12% -42%) to give the title compound as a white solid (80 mg, 26.1%).
Step 6:1- (((2S) -1- ((2- (6-amino-5- (8- (2- ((R) -4- (2- ((5- ((R) -1- ((2S, 4R) -4-hydroxy-2- (((S) -1- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) isoxazol-3-yl) oxy) ethyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) -4-fluorophenoxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylic acid
Figure BDA0004113777320001921
To a solution containing (2S, 4R) -4-hydroxy-1- ((R) -3-methyl-2- (3- (2-oxoethoxy) isoxazol-5-yl) butanoyl) -N- ((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) pyrrolidine-2-carboxamide (70.3 mg,0.13 mmol) and 1- (((2S) -1- ((4- ((2- (6-amino-5- (8- (2- ((R) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]Octane-3-yl) -4-fluorophenoxy-methyl) phenyl) -amino) -1-oxo-5-ureidopentan-2-yl carbamoyl) cyclobutanecarboxylic acid 2, 2-trifluoroacetate (80.0 mg,0.09 mmol) in dichloromethane (0.6 mL) and methanol (0.6 mL) was added sodium cyanoborohydride (11.1 mg,0.17 mmol) and sodium acetate (6.0 mg,0.07 mmol) as a drop of acetic acid. The reaction mixture was stirred at 20℃for 3 hours. The residue obtained was purified using Phenomenex Gemini-NX 80X 40mm X3 um (acetonitrile 17-47/0.05% NH) 3 H 2 O aqueous solution) to give the title compound (80 mg, yield 63.8%) as a white solid.
LCMS(ESI)m/z：1448.8[M+H] + 。
Step 7: n- ((2S) -1- ((4- ((2- (6-amino-5- (8- (2- (2- ((R) -4- (2- ((5- ((R) -1- ((2S, 4R) -4-hydroxy-2- (((S) -1- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) oxy) isoxazol-3-yl) oxy) ethyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) -4-fluorophenoxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) -N- (5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) cyclobutane-1, 1-dicarboxamide
Figure BDA0004113777320001931
To a mixture of 1- (((2S) -1- ((2- (6-amino-5- (8- (2- ((R) -4- (2- ((5- ((R) -1- ((2S, 4R) -4-hydroxy-2- (((S) -1- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) isoxazol-3-yl) oxy) ethyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) -pyridazin-3-yl) -4-fluorophenoxy) methyl) phenyl) -1-oxo-5-ureidopentan-2-yl) cyclobutanecarboxylic acid (80.0 mg,0.06 mmol) and 1- (5-aminopentyl) pyrrole-2, 5-dione; to a mixture of 2, 2-trifluoroacetic acid (19.6 mg,0.07 mmol) in N, N-dimethylformamide (4.00 mL) was added 2- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate (25.2 mg,0.07 mmol), N, N-diisopropylethylamine (0.03 mL,0.1700 mmol). The reaction mixture was stirred at 25 ℃ for 3 hours. The reaction mixture was concentrated to dryness by an oil pump. The residue was purified by preparative HPLC (Boston Green ODS 150 x 30mm x 5um, water (0.075% trifluoroacetic acid) -acetonitrile 20% -50%) to give the title compound (68.2 mg, 75%) as a white solid.
LCMS(ESI)m/z：806.7[M/2+H] + 。
1 H NMR(400MHz,DMSO-d 6 )δ(ppm)10.19(s,1H),9.02-8.86(m,1H),8.39(d,J＝8.0Hz,1H),7.94(d,J＝6.8Hz,1H),7.87-7.78(m,2H),7.65(d,J＝8.0Hz,2H),7.51-7.41(m,5H),7.36(d,J＝6.8Hz,5H),7.29-7.09(m,1H),6.97(s,2H),6.76(s,1H),6.42(s,1H),6.15-5.96(m,2H),5.07(s,2H),4.93-4.86(m,1H),4.71-4.60(m,2H),4.52-4.24(m,9H),3.66(s,4H),3.33(d,J＝7.2Hz,5H),3.09-2.96(m,8H),2.45(s,3H),2.42-2.34(m,4H),2.29-2.14(m,2H),2.04(d,J＝11.2Hz,3H),1.91(s,2H),1.83-1.55(m,5H),1.51-1.30(m,9H),1.19(d,J＝5.8Hz,5H),0.96(d,J＝6.4Hz,3H),0.86-0.75(m,3H)。
Synthesis example 17
Synthesis of L1-CIDE-BRM1-17
Figure BDA0004113777320001941
Step 1: (9H-fluoren-9-yl) methyl (2- ((hydroxyhydrophosphoryl) oxy) ethyl) carbamate
Figure BDA0004113777320001942
To a solution of (9H-fluoren-9-yl) methyl (2-hydroxyethyl) carbamate (1.0 g,3.53 mmol) in tetrahydrofuran (3.00 mL) was added tetrahydrofuran (5.0 mL) containing phosphorus trichloride (0.73 mL,8.44 mmol) and tetrahydrofuran (3.0 mL) containing triethylamine (1.1 mL,7.89 mmol) at-78 ℃. The reaction mixture was stirred at-78 ℃ for 20 minutes and then warmed to 25 ℃. The resulting mixture was stirred at 25℃for 12 hours. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (10 mL x 3). The organic phase was washed with brine (20 ml x 2), dried over sodium sulfate, filtered and concentrated to give the title compound (1.20 g, 97.9%) as a white solid.
LCMS(ESI)m/z：695.3[2M+H] + 。
Step 2: (9H-fluoren-9-yl) methyl (2- ((hydroxy (1H-imidazol-1-yl) phosphoryl) oxy) ethyl) carbamate
Figure BDA0004113777320001943
To a solution of (9H-fluoren-9-yl) methyl (2- ((hydroxyhydrophosphoryl) oxy) ethyl) carbamate (0.50 g,1.44 mmol) and triethylamine (0.6 mL,4.32 mmol) in carbon tetrachloride (5.0 mL) and acetonitrile (5.0 mL) was added 1- (trimethylsilyl) -1H-imidazole (0.61 g,4.32 mmol) at 25 ℃. The reaction mixture was stirred at 25 ℃ for 40 minutes. The mixture was treated with methanol (0.1 mL) and stirred at 25 ℃ for 10 min. The solvent was removed and the residue was washed with methyl tert-butyl ether/ethyl acetate=5/1 (3.0 mL), the precipitate was filtered and washed with tert-butyl ether (3.00 mL) to give the title compound (590 mg, 99.1% yield) as a yellow oil.
LCMS(ESI)m/z：414.3[M+H] + 。
Step 3:4- ((1 r,3 r) -3- ((4- (3- (3-amino-6- (2- (((di-tert-butoxyphosphoryl) oxy) methoxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) cyclobutoxy) piperidine-1-carboxylic acid tert-butyl ester
Figure BDA0004113777320001951
To a solution of tert-butyl 4- ((1 r,3 r) -3- ((4- (3- (3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) cyclobutoxy) piperidine-1-carboxylate (1.70 g,2.64 mmol) in N, N-dimethylformamide (36.0 mL) was added cesium carbonate (1.72 g,5.28 mmol) and di-tert-butyl chloromethyl phosphate (1.02 g,3.96 mmol). The reaction mixture was stirred at 70℃for 12 hours. The reaction mixture was quenched with water (150 mL) and extracted with ethyl acetate (80 mL x 3). The combined organic layers were washed with brine (100 ml x 2), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=100:1 to 50:1) to give the title compound (1.05 g, yield 45.9%) as a colorless oil.
LCMS(ESI)m/z：866.4[M+H] + 。
Step 4: (2- (6-amino-5- (8- (2- ((1 r,3 r) -3- (piperidin-4-yloxy) cyclobutoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) methyl dihydro-phosphate 2, 2-trifluoro-acetic acid
Figure BDA0004113777320001952
To a solution of tert-butyl 4- ((1 r,3 r) -3- ((4- (3-amino-6- (2- (((di-tert-butoxyphosphoryl) oxy) methoxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) cyclobutoxy) piperidine-1-carboxylate (1.05 g,1.21 mmol) in dichloromethane (36.0 mL) was added trifluoroacetic acid (0.09 mL,1.21 mmol). The reaction mixture was stirred at 20℃for 12 hours. The reaction was concentrated to give the title compound (930 mg, 99%) as a yellow oil.
LCMS(ESI)m/z：654.4[M+H] + 。
Step 5: (2- (6-amino-5- (8- (2- ((1R, 3R) -3- ((1- (2- ((5- ((R) -1- ((2S, 4R) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -4-hydroxypyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) isoxazol-3-yl) oxy) ethyl) piperidin-4-yl) oxy) cyclobutoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) phosphoric acid dihydro methyl ester
Figure BDA0004113777320001961
To a solution of (2S, 4R) -N- ((S) -1- (4-cyanophenyl) ethyl) -4-hydroxy-1- ((R) -3-methyl-2- (3- (2-oxoethoxy) isoxazol-5-yl) butanoyl) pyrrolidine-2-carboxamide (618 mg,1.21 mmol) in dichloromethane (0.6 mL) and methanol (0.6 mL) was added (2- (6-amino-5- (8- (2- ((1R, 3R) -3- (piperidin-4-yloxy) cyclobutoxy) pyridin-4-yl) -3, 8-diazabicyclo [ 3.2.1) ]Octane-3-yl) pyridazin-3-yl phenoxy) methyl 2, 2-trifluoro acetic acid (930 mg,1.21 mmol), sodium cyanoborohydride (155 mg,2.42 mmol) and sodium acetate (596 mg,7.26 mmol) as one drop of acetic acid. The reaction mixture was stirred at 20℃for 3 hours. The reaction was purified by preparative HPLC under the following conditions: chromatographic column, phenomenex Gemini-NX 80 x 40mm x 3um; mobile phase: 11-41% (water (0.05% NH) 3 H 2 O) -acetonitrile); detector, UV 254nm to give the title compound as a white solid (700 mg, 52.2% yield). LCMS (ESI) m/z:1106.5[ M+H ]] + 。
Step 6: (9H-fluoren-9-yl) methyl (2- (((((2- (6-amino-5- (8- (2- ((1R, 3R) -3- ((1- (2- ((5- ((R) -1- ((2S, 4R) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -4-hydroxypyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) isoxazol-3-yl) oxy) ethyl) piperidin-4-yl) oxy) cyclobutoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) methoxy) (hydroxy) phosphoryl) oxy) ethyl) carbamate
Figure BDA0004113777320001971
To a catalyst comprising (2- (6-amino-5- (8- (2- ((1R, 3R) -3- ((1- (2- ((5- ((R) -1- ((2S, 4R) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -4-hydroxypyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) isoxazol-3-yl) oxy) ethyl) piperidin-4-yl) oxy) cyclobutoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1 ]To a solution of methyl dihydrogen phosphate (200 mg,0.18 mmol) in N, N-dimethylformamide (36.0 mL) was added 1M zinc dichloride in tetrahydrofuran (1.45 mL,1.45 mmol) and methyl (9H-fluoren-9-yl) methyl (2- ((hydroxy (1H-imidazol-1-yl) phosphoryl) oxy) ethyl) carbamate (149 mg,0.36 mmol). The reaction mixture was stirred at 20℃for 12 hours. The crude product was purified by preparative HPLC under the following conditions: chromatographic column, phenomenex Gemini-NX 80 x 40mm x 3 um; mobile phase: 9-39% Water (0.05% NH) 3 H 2 O) -acetonitrile); the title compound was obtained as a white solid (200 mg, 76.2% yield) on a detector, UV 254 nm.
LCMS(ESI)m/z：726.7[M/2+H] + 。
Step 7: 2-Aminoethoxy (hydroxy) phosphoryl ] [2- [ 6-amino-5- [8- [2- [3- [ [1- [2- [5- [ rac- (1R) -2-methyl-1- [ rac- (2S, 4R) -4-hydroxy-2- [ rac- (1S) -1- (4-cyanophenyl) ethyl ] carbamoyl ] pyrrolidine-1-carbonyl ] propyl ] isoxazol-3-yl ] oxyethyl ] -4-piperidinyl ] oxy ] cyclobutoxy ] -4-pyridinyl ] -3, 8-diazabicyclo [3.2.1] oct-3-yl ] pyridazin-3-yl ] phenoxy ] hydrogen phosphate
Figure BDA0004113777320001972
To a (9H-fluoren-9-yl) -containing methyl group (2- ((((((2- (6-amino-5- (8- (2- ((1R, 3R) -3- ((1- (2- ((R) -1- ((2S, 4R) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -4-hydroxypyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) isoxazol-3-yl) oxy) ethyl) piperidin-4-yl) oxy) cyclobutoxy) pyridin-4-yl) -3, 8-diazabicyclo [ 3.2.1) ]Octane-3-yl) pyridazin-3-yl) phenoxy) methoxy (hydroxy) phosphoryl) oxy) ethyl) carbamate (200 mg,0.14 mmol) in N, N-dimethylformamide (10.0 mL) was added piperidine(0.10 mL,1.40 mmol). The reaction mixture was stirred at 20℃for 12 hours. Quench with 1N HCl (1.00 ml) and purify the resulting residue using Phenomenex Gemini-NX 80X 40mm X3 um (acetonitrile 19-49/water (0.05% NH) 3 H 2 O) -acetonitrile, 20-50%) to give the title compound (40.0 mg, 22.4% yield) as a white solid.
LCMS(ESI)m/z：1229.7[M+H] + 。
Step 8: (2S, 4R) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -4- (((1- (4- ((S) -2- (1- ((5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) carbamoyl) cyclobutanecarboxamide) amino) -5-ureidovaleryl) phenyl) -2- (4-methylpiperazin-1-yl) -2-oxoethoxy) carbonyl) oxy) pyrrolidine-1-carboxylic acid tert-butyl ester
Figure BDA0004113777320001981
To a mixture of methyl 2-aminoethoxy (hydroxy) phosphoryl ] [2- [ 6-amino-5- [8- [2- [3- [ [1- [2- [5- [ rac- (1R) -2-methyl-1- [ rac- (2S, 4R) -4-hydroxy-2- [ [ rac- (1S) -1- (4-cyanophenyl) ethyl ] carbamoyl ] pyrrolidin-1-carbonyl ] propyl ] isoxazol-3-yl ] oxyethyl ] -4-piperidinyl ] oxy ] cyclobutoxy ] -4-pyridinyl ] -3, 8-diazabicyclo [3.2.1] oct-3-yl ] pyridazin-3-yl ] phenoxy ] phosphate (120 mg,0.10 mmol) in anhydrous tetrahydrofuran (12.0 mL) was added N, N-diisopropylethylamine (18.7 uL,0.11 mmol) followed by 2, 5-dioxopyrrolidin-1-yl 6- (2, 5-dioxo-2, 5-dihydro-1H-1-pyrrol-1-yl) hexanoate (33.1 mg, 1 mmol). The reaction solution was stirred at 25℃for 16 hours. The solution was filtered and concentrated to dryness. The residue was purified by preparative HPLC (Boston Green ODS 150 x 30mm x 5um, water (0.075% trifluoroacetic acid) -acetonitrile 20% -50%) to give the title compound (60.8 mg, 36.8%) as a white solid.
LCMS(ESI)m/z：1423.0[M+H]+。
Synthesis example 18
Synthesis of L1-CIDE-BRM1-18
Figure BDA0004113777320001991
Step 1: (2S, 4R) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -4- (((4-nitrophenoxy) carbonyl) oxy) pyrrolidine-1-carboxylic acid tert-butyl ester
Figure BDA0004113777320001992
/>
To a mixture of tert-butyl (2S, 4R) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -4-hydroxypyrrolidine-1-carboxylate (1.00 g,2.78 mmol) in dichloromethane (10.0 mL) was added 2, 6-lutidine (0.49 mL,4.17 mmol) and 4-nitrophenyl chloroformate (673 mg,3.34 mmol). The reaction mixture was stirred at 25 ℃ for 18 hours. The crude mixture was concentrated to give the title compound (1.46 g, 36.8%) as a yellow solid. The crude product was immediately used in the next step.
LCMS(ESI)m/z：425.1[M-Boc+H] + 。
Step 2: (2S, 4R) -4- (((1- (4- ((S) -2- (1- ((allyloxy) carbonyl) cyclobutanecarboxamido) -5-ureidovaleramido) phenyl) -2- (4-methylpiperazin-1-yl) -2-oxoethoxy) carbonyl) oxy) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) pyrrolidine-1-carboxylic acid tert-butyl ester
Figure BDA0004113777320002001
To a mixture of tert-butyl (2S, 4 r) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -4- (((4-nitrophenoxy) carbonyl) oxy) pyrrolidine-1-carboxylate (1.46 g,2.78 mmol) and allyl (2S) -1- ((4- (1-hydroxy-2- (4-methylpiperazin-1-yl) -2-oxoethyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutanecarboxylate (1.59 g,2.78 mmol) in N, N-dimethylformamide (15.0 mL) was added 4-dimethylaminopyridine (680 mg,5.56 mmol). The reaction mixture was stirred at 25 ℃ for 18 hours. The crude product was filtered and purified by preparative HPLC (Phenomenex Gemini-NX 80 x 30mM x 3 um/water (10 mM NH 4 HCO 3 ) Acetonitrile/10%)80%) to give the title compound as a yellow solid (400 mg, 15%). LCMS (ESI) m/z:958.5[ M+H ]] + 。
Step 3:1- (((2S) -1- ((4- (1- ((((((3 r, 5S) -1- (tert-butoxycarbonyl) -5- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) carbonyl) oxy) -2- (4-methylpiperazin-1-yl) -2-oxoethyl) phenyl) amino) -1-oxo-5-ureidopent-2-yl) carbamoyl) cyclobutanecarboxylic acid
Figure BDA0004113777320002002
To a solution of (2S, 4 r) -4- (((1- (4- ((S) -2- (1- ((allyloxy) carbonyl) cyclobutanecarboxamido) -5-ureidopentanoylamino) phenyl) -2- (4-methylpiperazin-1-yl) -2-oxoethoxy) carbonyl) oxy) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) pyrrolidine-1-carboxylic acid tert-butyl ester (260 mg,0.27 mmol) and 1, 3-dimethylpyrimidine-2, 4,6 (1 h,3h,5 h) -trione (212 mg,1.36 mmol) in dichloromethane (2.00 mL) and methanol (2.00 mL) was added tetrakis (triphenylphosphine) palladium (62.7 mg,0.05 mmol) at 25 ℃. The reaction mixture was stirred under nitrogen at 25 ℃ for 16 hours. The crude product was concentrated and purified by preparative HPLC under the following conditions: column: phenomenex Gemini-NX 80 x 30mm x 3um, mobile phase: water (10 mM NH) 4 HCO 3 ) Acetonitrile 10% -80% to give the title compound (110 mg, 44.2% yield) as a yellow solid.
LCMS(ESI)m/z：918.6[M+H] + 。
Step 4: (2S, 4R) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -4- (((1- (4- ((S) -2- (1- ((5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) carbamoyl) cyclobutanecarboxamide) amino) -5-ureidovaleryl) phenyl) -2- (4-methylpiperazin-1-yl) -2-oxoethoxy) carbonyl) oxy) pyrrolidine-1-carboxylic acid tert-butyl ester
Figure BDA0004113777320002011
To a liquid containing 1- (((2S) -1- ((4- (1- ((((3R, 5S))To a mixture of (1- (tert-butoxycarbonyl) -5- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) pyrrolidin-3-yl) oxy) carbonyl) oxy) -2- (4-methylpiperazin-1-yl) -2-oxoethyl) phenyl) -1-oxo-5-ureidopent-2-yl carbamoyl) cyclobutanecarboxylic acid (110 mg,0.12 mmol) and 1- (5-aminopentyl) -1H-pyrrole-2, 5-dione 2, 2-trifluoroacetic acid (43.0 mg,0.15 mmol) in N, N-dimethylformamide (3 mL) was added N, N-diisopropylethylamine (0.06 mL,0.36 mmol) and 2- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate (54.7 mg,0.14 mmol). The reaction mixture was stirred at 25℃for 3h. The mixture was concentrated by an oil pump. The residue was purified by preparative HPLC (Boston Green ODS 150X 30mm X5 um, water (0.075% TFA) -acetonitrile 28% -58%) to give the title compound (90 mg, 69.4%) as a white solid. LCMS (ESI) m/z:1082.6[ M+H ] ] + 。
Step 5: (3R, 5S) -5- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) pyrrolidin-3-yl (1- (4- ((S) -2- (1- ((5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) carbamoyl) cyclobutanecarboxamido) -5-ureidovaleryl) phenyl) -2- (4-methylpiperazin-1-yl) -2-oxoethyl) carbonate 2, 2-trifluoroacetate
Figure BDA0004113777320002021
To a solution of tert-butyl (2S, 4 r) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -4- (((1- (4- ((S) -2- (1- ((5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) carbamoyl) cyclobutanecarboxamido) -5-ureidopentanoylamino) phenyl) -2- (4-methylpiperazin-1-yl) -2-oxoethoxy) carbonyl) oxy) pyrrolidine-1-carboxylate (90 mg,0.08 mmol) in hexafluoroisopropanol (5 mL) containing 5% trifluoroacetic acid was stirred at 25 ℃ for 2 hours. The mixture was concentrated to give the title compound (91.0 mg, yield 99.8%) as a yellow oil. LCMS (ESI) m/z:982.4[ M-TFA+H] + 。
Step 6: (3R, 5S) -1- (2- (3- (2- (4- ((1 r,3 r) -3- ((4- (3- (3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) oxy) cyclobutoxy) piperidin-1-yl) ethoxy) isoxazol-5-yl) -3-methylbutanoyl) -5- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) pyrrolidin-3-yl (1- (4- ((S) -2- (1- ((5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) carbamoyl) cyclobutanecarboxamido) -5-ureidovaleryl) phenyl) -2- (4-methylpiperazin-1-yl) -2-oxoethyl) carbonate
Figure BDA0004113777320002022
To a catalyst containing (3R, 5S) -5- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) pyrrolidin-3-yl (1- (4- ((S) -2- (1- ((5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) carbamoyl) cyclobutanecarboxamido) -5-ureidopentanamino) phenyl) -2- (4-methylpiperazin-1-yl) -2-oxoethyl) carbonate 2, 2-trifluoroacetate (91.0 mg,0.08 mmol) and 2- (3- (2- ((1 r,3 r) -3- ((4- (3- (3-amino-6- (2-hydroxyphenyl) pyridazin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]To a mixture of N, N-dimethylformamide (2.50 mL) with N, N-diisopropylethylamine (0.08 mL,0.50 mmol) and 2- (7-azabenzotriazol-1-yl) -N, N' -tetramethylurea hexafluorophosphate (41.0 mg,0.11 mmol) was added octan-8-yl-pyridin-2-yloxy) cyclobutoxy) piperidin-1-yl-ethoxy) isoxazol-5-yl) -3-methylbutanoic acid (81.5 mg,0.11 mmol). The mixture was stirred at 25℃for 16 hours. After concentration, the crude product was purified by preparative HPLC under the following conditions: column: welch xomate C18.40 mm.3 um (15-45% over water (0.075% trifluoroacetic acid) -acetonitrile) to give the title compound (80.6 mg, 52% yield) as a white solid. LCMS (ESI) m/z:860.4[1/M+H] +
Intermediate 1: (S) -1- ((1- ((4- ((2-bromophenoxy) methyl) phenyl) amino) -6- (dimethylamino) -1-oxohex-2-yl) carbamoyl) cyclobutane-1-carboxylic acid ethyl ester
Figure BDA0004113777320002031
Step 1: n (N) 2 - (tert-butoxycarbonyl) -N 6 ,N 6 -dimethyl-L-lysine
Figure BDA0004113777320002032
Under hydrogen (3 atm), the mixture containing (t-butoxycarbonyl) -L-lysine (20.0 g,81.2 mmol), CH 2 A solution of O (12.2 g,162 mmol) and Pd/C (2.00 g) in methanol (100 mL) was stirred at room temperature for 4 hours. After filtration, the filtrate was concentrated under reduced pressure. The residue was taken up in Et 2 And (3) washing. The solid was collected by filtration to give 20.6g (92% yield) of the title compound as a white solid. LCMS (ESI) [ M+H ]] + =275. Step 2: 1-bromo-2- ((4-nitrobenzyl) oxy) benzene
Figure BDA0004113777320002041
2-bromophenol (52.6 g,304 mmol), 1- (bromomethyl) -4-nitrobenzene (65.7 g,304 mmol) and K 2 CO 3 A solution of (83.9 g,608 mmol) in DMF (700 mL) was stirred at room temperature for 1 hour. EtOAc was added and washed 3 times with water. The organic layer was treated with anhydrous Na 2 SO 4 Dried and concentrated in vacuo to give 73.8g (78% yield) of the title compound as a yellow solid. 1 H NMR(300MHz,DMSO-d6)δ8.34–8.24(m,2H),7.81–7.70(m,2H),7.62(dd,J＝7.9,1.6Hz,1H),7.36(ddd,J＝8.3,7.3,1.6Hz,1H),7.20(dd,J＝8.3,1.5Hz,1H),6.94(td,J＝7.6,1.4Hz,1H),5.39(s,2H)。
Step 3:4- ((2-bromophenoxy) methyl) aniline
Figure BDA0004113777320002042
To a mixture of 1-bromo-2- ((4-nitrobenzyl) oxy) benzene (43.0 g,139.5 mmol) and K under nitrogen at 0deg.C 2 CO 3 Na was added in portions to a solution of (115 g,837 mmol) in acetonitrile (800 mL) and water (400 mL) 2 S 2 O 4 (242 g,1395 mmol). The mixture was stirred at room temperature for 6 hours. The product was extracted once with EtOAc. The organic layer was treated with anhydrous Na 2 SO 4 Dried and concentrated in vacuo to give 35g (crude) of the title compound as a yellow solidAnd (3) a compound. LCMS (ESI) [ M+H ]] + ＝278。
Step 4: (S) - (1- ((4- ((2-bromophenoxy) methyl) phenyl) amino) -6- (dimethylamino) -1-oxohex-2-yl) carbamic acid tert-butyl ester
Figure BDA0004113777320002043
Under nitrogen at 25 ℃ to N-containing 2 - (tert-butoxycarbonyl) -N 6 ,N 6 To a solution of dimethyl-L-lysine (13.3 g,48.4 mmol) and NMM (10.3 g,96.9 mmol) in tetrahydrofuran (200 mL) was added isobutyl-chloroformate (7.91 g,58.1 mmol) dropwise. The reaction was stirred at 25℃for 0.5 h. A solution of 4- ((2-bromophenoxy) methyl) aniline (16.1 g, crude) in tetrahydrofuran (120 mL) was then added at-25 ℃. The reaction mixture was stirred at room temperature for 4 hours. The solvent was concentrated under vacuum. DCM was added and washed with water. The organic layer was treated with anhydrous Na 2 SO 4 Dried, and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (gradient: 0-9% MeOH/DCM) to give 6.70g (25% yield) of the title compound as a white solid. LCMS (ESI) [ M+H ]] + ＝534。
Step 5: (S) -2-amino-N- (4- ((2-bromophenoxy) methyl) phenyl) -6- (dimethylamino) hexanamide (2, 2-trifluoroacetate salt)
Figure BDA0004113777320002051
A solution of tert-butyl (S) - (1- ((4- ((2-bromophenoxy) methyl) phenyl) amino) -6- (dimethylamino) -1-oxohex-2-yl) carbamate (4.00 g,7.48 mmol) in 5% TFA/HFIP (50 mL) was stirred at room temperature for 3 hours. The solvent was concentrated in vacuo and used directly in the next step. LCMS (ESI) [ M+H ] ] + ＝434。
Step 6: (S) -1- ((1- ((4- ((2-bromophenoxy) methyl) phenyl) amino) -6- (dimethylamino) -1-oxohex-2-yl) carbamoyl) cyclobutane-1-carboxylic acid ethyl ester
Figure BDA0004113777320002052
To a solution of (S) -2-amino-N- (4- ((2-bromophenoxy) methyl) phenyl) -6- (dimethylamino) hexanamide (2, 2-trifluoroacetate) (crude product from step 5), 1- (ethoxycarbonyl) cyclobutane-1-carboxylic acid (1.55 g,8.98 mmol) and DIPEA (9.65 g,74.8 mmol) in DMF (20 mL) was added HATU (3.41 g,8.98 mmol) at 0deg.C. The mixture was stirred at room temperature for 0.5 hours. The crude product was purified using a pre-packed C18 column (gradient: 0-100% MeOH in water (0.05% nh) 4 HCO 3 ) To obtain 2.70g (yield 61%) of the title compound as a red solid. LCMS (ESI) [ M+H ]] + ＝588。 1 H NMR(300MHz,DMSO-d6)δ9.87(s,1H),7.72(d,J＝7.9Hz,1H),7.54–7.42(m,3H),7.30(d,J＝8.6Hz,2H),7.21(ddd,J＝8.8,7.3,1.6Hz,1H),7.07(dd,J＝8.4,1.5Hz,1H),6.77(td,J＝7.6,1.4Hz,1H),5.02(s,2H),4.30(q,J＝8.0Hz,1H),4.00(q,J＝7.1Hz,2H),2.35–2.21(m,2H),2.03(t,J＝6.8Hz,2H),1.96(s,6H),1.80-1.41(m,4H),1.30-1.14(m,6H),1.06(t,J＝7.1Hz,3H)。
Intermediate 5: (3R) -4- (2- ((4- (3- (3-amino-6- (2- (methoxymethoxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester
Figure BDA0004113777320002061
Step 1: (3R) -4- (2- ((4- (3- (3-amino-6- (2- (methoxymethoxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester
Figure BDA0004113777320002062
(3R) -4- (2- ((4- (3- (3-amino-6-chloropyridazin-4-yl) -3, 8-diazabicyclo [ 3.2.1) at 100 ℃ C.) under nitrogen ]Octan-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazine-1-carboxylic acid tert-butyl esterButyl ester (1.00 g,1.79 mmol), (2- (methoxymethoxy) phenyl) organoboronic acid (399mg, 2.15 mmol), pd (PPh) 3 ) 4 (413 mg, 0.356 mmol) and K 2 CO 3 A solution of (741mg, 5.37 mmol) dioxane (10 mL) and water (2 mL) was stirred for 1 hour. The reaction was diluted with water and extracted with dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (gradient: 0% -10% methanol/dichloromethane) to give 670mg (yield 57%) of the title compound as a yellow solid. LC-MS: (ESI, M/z) [ M+H ]] + ＝661。 1 H NMR(300MHz,DMSO-d 6 )δ7.76(d,J＝5.9Hz,1H),7.58(dd,J＝7.6,1.8Hz,1H),7.34(ddd,J＝9.0,7.3,1.8Hz,1H),7.18–7.01(m,3H),6.51(dd,J＝6.1,2.0Hz,1H),6.12(d,J＝2.0Hz,1H),5.72(s,2H),5.14(s,2H),4.47(s,2H),4.25(t,J＝6.1Hz,2H),3.53(d,J＝12.8Hz,2H),3.22(s,3H),3.14–2.67(m,8H),2.64-2.56(m,1H),2.46-2.36(m,1H),2.32-2.22(m,1H),2.22-2.13(m,2H),2.00-1.90(m,2H),1.38(s,9H),0.96(d,J＝6.2Hz,3H)。
Synthesis example 19
Synthesis of L1-CIDE-BRM1-19
Figure BDA0004113777320002071
Step 1: (S) -1- ((6- (dimethylamino) -1-oxo-1- ((4- ((2- (4, 5-tetramethyl-1, 3, 2-dioxapentaborane-2-yl) phenoxy) methyl) phenyl) amino) hexane-2-yl) carbamoyl) cyclobutane-1-carboxylic acid ethyl ester
Figure BDA0004113777320002072
(S) -1- ((1- ((4- ((2-bromophenoxy) methyl) phenyl) amino) -6- (dimethylamino) -1-oxohex-2-yl) carbamoyl) cyclobutane-1-carboxylic acid ethyl ester (500 mg,0.852 mmol), B) at 80℃under nitrogen 2 Pin 2 (649mg,2.55mmol)、Pd(dppf)Cl 2 (124 mg,0.170 mmol) and KOAc (250 mg,2.55 mmol) in 1, 4-dioxane (5 mL) were stirred for 2 hours. The reaction is carried out Dilute with DCM and wash with water. The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (gradient: 0% -20% MeOH/DCM (containing 0.3%7 MNH) 3 MeOH) to yield 390mg (72% yield) of the title compound as a yellow solid. LC-MS: (ESI, M/z) [ M+H ]] + ＝636。
Step 2:4- ((1 r,3 r) -3- ((4- (3-amino-6- (2- ((4- ((S) -6- (dimethylamino) -2- (1- (ethoxycarbonyl) cyclobutane-1-carboxamido) hexanamido) benzyl) oxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) cyclobutoxy) piperidine-1-carboxylic acid tert-butyl ester
Figure BDA0004113777320002081
Under nitrogen, a mixture of (S) -1- ((6- (dimethylamino) -1-oxo-1- ((4- ((2- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenoxy) methyl) phenyl) amino) hexane-2-yl) carbamoyl) cyclobutane-1-carboxylic acid ethyl ester (390 mg,0.614 mmol), 4- ((1 r,3 r) -3- ((4- (3- (3-amino-6-chloropyridazin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]Octan-8-yl) pyridin-2-yl) oxy) cyclobutoxy) piperidine-1-carboxylic acid tert-butyl ester (399mg, 0.676 mmol), ad 2 nBuPPdG2 (41.0 mg,0.061 mmol) and K 2 CO 3 (260 mg,1.22 mmol) dioxane (5.0 mL) and H 2 A solution of O (1.2 mL) was stirred for 2 hours. The resulting solution was diluted with water and extracted with EtOAc. The organic layer was concentrated under vacuum. The residue was purified using a pre-packed C18 column (solvent gradient: 0-100% meoh in water (0.1% NH) 4 HCO 3 ) To give 310mg (yield 47%) of the title compound as a white solid. LC-MS: (ESI, M/z) [ M+H ]] + ＝1060。
Step 3:1- (((2S) -1- ((4- ((2- (6-amino-5- (8- (2- ((1 r,3 r) -3- ((1- (tert-butoxycarbonyl) piperidin-4-yl) oxy) cyclobutoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) methyl) phenyl) amino) -6- (dimethylamino) -1-oxohex-2-yl) carbamoyl) cyclobutane-1-carboxylic acid lithium
Figure BDA0004113777320002082
Will contain 4- ((1 r,3 r) -3- ((4- (3-amino-6- (2- ((4- ((S) -6- (dimethylamino) -2- (1- (ethoxycarbonyl) cyclobutane-1-carboxamide) hexanamido) benzyl) oxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1]Octane-8-yl) pyridin-2-yl) oxy) cyclobutoxy) piperidine-1-carboxylic acid tert-butyl ester (270 mg,0.255 mmol) and LiOH. H 2 THF (2 mL) and H for O (32.1 mg,0.765 mmol) 2 A solution of O (2 mL) was stirred at room temperature for 1 hour. The resulting mixture was concentrated under vacuum. The product was used directly in the next step. LC-MS: (ESI, M/z) [ M+H ]] + ＝1032。
Step 4:1- (((2S) -1- ((4- ((2- (6-amino-5- (8- (2- ((1 r,3 r) -3- (piperidin-4-yloxy) cyclobutoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) methyl) phenyl) amino) -6- (dimethylamino) -1-oxohex-2-yl) carbamoyl) cyclobutane-1-carboxylic acid
Figure BDA0004113777320002091
Will contain 1- (((2S) -1- ((4- ((2- (6-amino-5- (8- (2- ((1 r,3 r) -3- ((1- (tert-butoxycarbonyl) piperidin-4-yl) oxy) cyclobutoxy) pyridin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]A solution of lithium octan-3-yl) pyridazin-3-yl-phenoxy-methyl) phenyl) -6- (dimethylamino) -1-oxohex-2-yl-carbamoyl) cyclobutane-1-carboxylate (crude product from the previous step) in TFA (0.75 mL) and HFIP (14 mL) was stirred at room temperature for 30 min. The resulting mixture was concentrated under vacuum. The resulting crude product was purified using a pre-packed C18 column (solvent gradient: 0-100% MeOH in water (0.1% nh) 4 HCO 3 ) To give 170mg (71% yield) of the title compound as a yellow solid. LC-MS: (ESI, M/z) [ M+H ]] + ＝932。
Step 5:1- (((2S) -1- ((4- ((2- (6-amino-5- (8- (2- ((1R, 3R) -3- ((1- (2- ((5- ((R) -1- ((2S, 4R) -1- ((1- (4-cyanophenyl) ethyl) carbamoyl) -4-hydroxypyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) oxy) isoxazol-3-yl) oxy) piperidin-4-yl) oxy) cyclobutoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) methyl) phenyl) -6- (dimethylamino) -1-oxohex-2-yl) carbamoyl) cyclobutane-1-carboxylic acid
Figure BDA0004113777320002092
Under nitrogen, a composition containing 1- (((2S) -1- ((4- ((2- (6-amino-5- (8- (2- ((1 r,3 r) -3- (piperidin-4-yloxy) cyclobutoxy) pyridin-4-yl) -3, 8-diazabicyclo [ 3.2.1) ]Octane-3-yl) pyridazin-3-yl) phenoxy methyl) amino) -6- (dimethylamino) -1-oxohex-2-yl) carbamoyl) cyclobutane-1-carboxylic acid (170.0 mg, 0.183mmol), (2S, 4R) -N- ((S) -1- (4-cyanophenyl) ethyl) -4-hydroxy-1- ((R) -3-methyl-2- (3- (2-oxoethoxy) isoxazol-5-yl) butyryl) pyrrolidine-2-carboxamide (111 mg,0.237 mmol) and HOAc (21.9 mg,0.365 mmol) in DCM (1.5 mL) and MeOH (0.5 mL) were stirred at room temperature for 1 hour. Then NaBH is added at 0 DEG C 3 CN (17.3 mg,0.274 mmol) and was stirred at room temperature for 30 minutes. The reaction was quenched with water. The resulting solution was concentrated under vacuum. The crude product obtained was purified using a pre-packed C18 column (gradient: 0-100% MeOH in water (0.05% NH) 4 HCO 3 ) 180mg (71% yield) of the title compound was obtained as a white solid. LC-MS: (ESI, M/z) [ M+H ]] + ＝1384。
Step 5: n- ((2S) -1- ((4- ((2- (6-amino-5- (8- (2- ((1R, 3R) -3- ((1- (2- ((5- ((R) -1- ((2S, 4R) -1- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -4-hydroxypyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) oxy) ethyl) piperidin-4-yl) oxy) cyclobutoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) methyl) amino) -6- (dimethylamino) -1-oxohex-2-yl) -N- (5- (2, 5-dihydro-1H-pyrrol-1-yl) pentyl) cyclobutane-1, 1-dicarboxamide (formate salt)
Figure BDA0004113777320002101
To a solid containing 1- (((2S) -1- ((2- (6-amino-5- (8- (2- ((1R, 3R) -3- ((1- (2- ((5- ((R) -1- ((2S, 4R) -2- (((S) -1- (4-cyanophenyl) ethyl) carbamoyl) -4-hydroxypyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) isoxazol-3-yl) oxy) ethyl) piperidin-4-yl) oxy) cyclobutoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1 at room temperature]Octane-3-yl) pyridazin-3-yl-phenoxy-methyl) phenyl) -6- (dimethylamino) -1-oxohex-2-yl-carbamoyl) cyclobutane-1-carboxylic acid (65.0 mg,0.047 mmol), 1- (5-aminopentyl) -1H-pyrrole-2, 5-dione (2, 2-trifluoroacetate) (25.6 mg, crude product) and DIPEA (90.9 mg, 0.704 mmol) in DMF (1.5 mL) was added HATU (21.4 mg,0.056 mmol). The resulting solution was stirred at room temperature for 30 minutes. The resulting solution was passed through a preparative HPLC (Xselect CSH F-phenyl OBD column, 19X 250mm 5 μm; mobile phase A: water (0.05% FA), mobile phase B: ACN; flow rate: 60mL/min; gradient: increase from 2% B to 29% B;254nm R in 7 minutes) T1 6.5 min)) to give 5.9mg (yield 8%) of L1-CIDE-BRM1-19 as a white solid. LC-MS: (ESI, M/z) [ M+H ]] + ＝1548。 1 H NMR(300MHz,DMSO-d 6 )δ10.24(s,1H),δ8.47(d,J＝7.4Hz,1H),8.17(s,1H),7.93–7.54(m,8H),7.55-7.30(m,5H),7.26–7.09(m,2H),7.08–6.87(m,3H),6.43–5.88(m,3H),5.59(s,2H),5.22–4.86(m,5H),4.50–4.12(m,8H),3.72–3.54(m,3H),3.13–2.97(m,4H),2.63(s,6H),2.45-2.35(m,5H),2.25-2.02(m,16H),2.02–1.56(m,11H),1.57–1.25(m,13H),1.29-1.12(m,4H),0.95(d,J＝6.8Hz,3H),0.79(d,J＝6.8Hz,3H)。
Synthesis example 20
Synthesis of L1-CIDE-BRM1-20
4- ((S) -2- (1- ((5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) carbamoyl) cyclobutan-1-oylamino) -5-ureidovalerylamino) benzyl (4- (8- (2- ((R) -4- (2- ((5- ((R) -1- ((2S, 4R) -4-hydroxy-2- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) isoxazol-3-yl) oxy) ethyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] octane-3-yl) -6- (2-hydroxyphenyl) pyridazin-3-yl) carbamate (formate salt)
Figure BDA0004113777320002111
Step 1: (3R) -4- (2- ((4- (3- ((((4- ((S) -2- (1- (ethoxycarbonyl) cyclobutane-1-carboxamide) -5-ureidopentanoylamino) benzyl) oxy) carbonyl) amino) -6- (2- (methoxymethoxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester
Figure BDA0004113777320002112
To a nitrogen containing (3R) -4- (2- ((4- (3- (3-amino-6- (2- (methoxymethoxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [ 3.2.1) at 0deg.C]To a solution of tert-butyl octan-8-yl) pyridin-2-yloxy) -3-methylpiperazine-1-carboxylate (500 mg, 0.751 mmol, provided by Genetech), (S) -1- ((1- ((4- (hydroxymethyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutane-1-carboxylate (986 mg,2.26mmol, provided by Genetech) and DIPEA (488 mg,3.78 mmol) in THF (25 mL) was added triphosgene (85.5 mg, 0.284 mmol). The reaction was stirred at room temperature for 0.5 hours. The solvent was evaporated and the residue purified by flash chromatography on silica gel (gradient: 0% -13% methanol/dichloromethane) followed by purification through a pre-packed C18 column (solvent gradient: 0-100% acn in water (0.05% NH) 4 HCO 3 ) 191mg (22%) of the title compound were obtained as a white solid. LC-MS: (ESI, M/z) [ M+H ] ] + ＝1122。
Step 2:1- (((2S) -1- ((4- ((((4- (8- (2- (2- ((R) -4- (tert-butoxycarbonyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) -6- (2- (methoxymethoxy) phenyl) pyridazin-3-yl) carbamoyl) oxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutane-1-carboxylic acid lithium
Figure BDA0004113777320002121
(3R) -4- (2- ((4- (3- ((((4- ((S) -2- (1- (ethoxycarbonyl) cyclobutane-1-carboxamido) -5-ureidopentanoylamino) benzyl) oxy) carbonyl) amino) -6- (2- (methoxymethoxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]A solution of tert-butyl octan-8-yl) pyridin-2-yloxy) -ethyl-3-methylpiperazine-1-carboxylate (130 mg,0.115 mmol) and LiOH (14.0 mg,0.350 mmol) in THF (3 mL) and water (3 mL) was stirred at room temperature for 1 hour. THF was removed in vacuo and then lyophilized to give 140mg (crude) of the title compound as a white solid. LC-MS: (ESI, M/z) [ M+H ]] + ＝1094。
Step 3:1- (((2S) -1- ((4- ((((6- (2-hydroxyphenyl) -4- (8- (2- (2- ((R) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) carbamoyl) oxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutane-1-carboxylic acid
Figure BDA0004113777320002131
Under nitrogen, a composition containing 1- (((2S) -1- ((4- ((((4- (8- (2- ((R) -4- (tert-butoxycarbonyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]A solution of lithium octan-3-yl) -6- (2- (methoxymethoxy) phenyl) pyridazin-3-yl-carbamoyl) oxy) ethyl) phenyl) -1-oxo-5-ureidopentan-2-yl-carbamoyl) cyclobutane-1-carboxylate (240 mg,0.219 mmol) in concentrated HCl (2 mL), THF (2 mL) and isopropanol (2 mL) was stirred at room temperature for 0.5 h. The reaction solution was concentrated under reduced pressure, and the remaining aqueous solution was purified by passing through a pre-packed C18 column (solvent gradient: 0-100% ACN aqueous solution (0.05% NH) 4 HCO 3 ) To yield 150mg of the title compound as a yellow solid. LC-MS: (ESI, M/z) [ M+H ]] + ＝949。
Step 4:1- (((2S) -1- ((4- ((((4- (2- (2- ((R) -4- (2- ((5- ((R) -1- ((2S, 4R) -4-hydroxy-2- (((S) -1- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) oxy) isoxazol-3-yl) oxy) ethyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) -6- (2-hydroxyphenyl) pyridazin-3-yl) carbamoyl) oxy) methyl) phenyl) -1-oxo-5-ureidopentan-2-yl) carbamoyl) cyclobutane-1-carboxylic acid
Figure BDA0004113777320002132
Under nitrogen, a composition containing 1- (((2S) -1- ((4- ((((6- (2-hydroxyphenyl) -4- (8- (2- (2- ((R) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]Octane-3-yl) pyridazin-3-yl) carbamoyl) oxy) methyl) amino) -1-oxo-5-ureidon-2-yl-carbamoyl) cyclobutane-1-carboxylic acid (150 mg,0.158 mmol), (2S, 4R) -4-hydroxy-1- ((R) -3-methyl-2- (3- (2-oxoethoxy) isoxazol-5-yl) butyryl) -N- ((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) pyrrolidine-2-carboxamide (112 mg,0.207 mmol), CH 3 A solution of COOH (19.8 mg, 0.399 mmol) in methanol (3 mL) and DCM (1 mL) was stirred at 30deg.C for 1 hour. Then NaBH is added 3 CN (19.5 mg,0.513 mmol) was stirred at 30℃for 0.5 h. The reaction solution was concentrated under vacuum. The residue was purified using a pre-packed C18 column (solvent gradient: 0-100% aqueous methanol (0.05% NH) 4 HCO 3 ) 180mg (77%) of the title compound are obtained as a yellow solid. LC-MS: (ESI, M/z) [ M+H ]] + ＝1474。
Step 5:4- ((S) -2- (1- ((5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) carbamoyl) cyclobutan-1-oylamino) -5-ureidovalerylamino) benzyl (4- (8- (2- ((R) -4- (2- ((5- ((R) -1- ((2S, 4R) -4-hydroxy-2- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) isoxazol-3-yl) oxy) ethyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] octane-3-yl) -6- (2-hydroxyphenyl) pyridazin-3-yl) carbamate (formate salt)
Figure BDA0004113777320002141
To a solid containing 1- (((2S) -1- ((4- ((((4- (2- (2- ((R) -4- (2- ((R) -1- ((2S, 4R) -4-hydroxy-2- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) isoxazol-3-yl) oxy) ethyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1 at room temperature]Octane-3-yl) -6- (2-hydroxyphenyl) pyridazin-3-yl-carbamoyl) oxy) methyl) phenyl) -1-oxo-5-ureidopentan-2-yl-carbamoyl) cyclobutane-1-carboxylic acid (180 mg,0.122 mmol), 1- (5-aminopentyl) -1H-pyrrole-2, 5-dione (2, 2-trifluoroacetate) (67 mg, crude product) and DIPEA (158 mg,1.22 mmol) in DMF (3 mL) was added HATU (67.0 mg,0.176 mmol). The reaction was stirred at room temperature for 1 hour. The resulting solution was purified by preparative HPLC (column XBridge Prep OBD C column, 30X 150mm,5 μm; mobile phase A: water (0.1% FA), mobile phase B: ACN; flow rate: 60mL/min; gradient: increase from 8% B to 38% B over 7 min; wavelength: 254nm R T1 (min: 6.5 min)), to obtain 49.7mg (yield: 24.0%) of L1-CIDE-BRM1-20 as a white solid. LC-MS: (ESI, M/z) [ M+H ]] + ＝1638。 1 H NMR(300MHz,DMSO-d 6 )δ13.39(s,1H),10.13(s,1H),9.94(s,1H),8.99(s,1H),8.40(d,J＝7.7Hz,1H),8.14(s,1H),8.03(d,J＝7.9Hz,1H),7.82(dd,J＝8.0,5.8Hz,3H),7.70–7.61(m,3H),7.51–7.41(m,2H),7.41–7.28(m,5H),6.96(d,J＝16.1Hz,4H),6.54(d,J＝6.1Hz,1H),6.17(s,1H),6.10(s,1H),5.96(dd,J＝10.3,4.5Hz,1H),5.41(s,2H),5.09(d,J＝5.3Hz,3H),4.91(t,J＝7.1Hz,1H),4.50–4.34(m,4H),4.32-4.28(m,5H),3.74–3.61(m,2H),3.55-3.40(m,4H),3.39-3.35(m,3H),3.19–2.89(m,9H),2.70-2.60(m,2H),2.48-2.38(m,9H),2.23-2.12(m,1H),2.10-1.95(m,2H),1.88(s,4H),1.80-1.70(m,4H),1.68–1.57(m,1H),1.52-1.30(m,10H),1.28–1.16(m,2H),1.10-0.90(m,6H),0.82(d,J＝6.6Hz,3H)。
Synthesis example 21
Synthesis of L1-CIDE-BRM1-21
N- ((2S) -1- ((4- ((2- (6-amino-5- (8- (2- (2- ((R) -4- (2- ((5- ((R) -1- ((2S, 4R) -4-hydroxy-2- (((S) -1- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) oxy) isoxazol-3-yl) oxy) ethyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) methyl) amino) -6- (dimethylamino) -1-oxohex-2-yl) -N- (5- (2, 5-dioxo) -2, 5-dihydro-1H-pyrrol-1-yl) pentyl) cyclobutane-1, 1-dicarboxamide; 2, 2-trifluoro acetic acid
Figure BDA0004113777320002161
Step 1: (3R) -4- (2- ((4- (3-amino-6- (2- ((4- ((S) -6- (dimethylamino) -2- (1- (ethoxycarbonyl) cyclobutane-1-carboxamido) hexanamido) benzyl) oxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester
Figure BDA0004113777320002162
(S) -1- ((6- (dimethylamino) -1-oxo-1- ((4- ((2- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenoxy) methyl) phenyl) amino) hexane-2-yl) carbamoyl) cyclobutane-1-carboxylic acid ethyl ester (316 mg,0.570 mmol), (3R) -4- (2- ((4- (3-amino-6-chloropyridazin-4-yl) -3, 8-diazabicyclo [ 3.2.1) at 95℃under nitrogen ]Octan-8-yl) pyridin-2-yl) oxy) ethyl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester (538.8 mg,0.850 mmol), K 3 PO 4 (240 mg,1.13 mmol) and Ad 2 A solution of nBuPPdG2 (37.8 mg,0.0600 mmol) in 1, 4-dioxane (4 mL) and water (1 mL) was stirred for 3 hours. Water was added and extracted 3 times with EtOAc. The organic solvents were combined and concentrated in vacuo. The residue was purified using a pre-packed C18 column (solvent gradient: 0-100% ACN in water (0.05% NH) 4 HCO 3 ) The residue was purified to give 230mg (39% yield) of the title compound as a red solid. LCMS (ESI) [ M+H ]] + ＝1032
Step 2:1- (((2S) -1- ((4- ((2- (6-amino-5- (8- (2- (2- ((R) -4- (tert-butoxycarbonyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) methyl) phenyl) amino) -6- (dimethylamino) -1-oxohex-2-yl) carbamoyl) cyclobutane-1-carboxylic acid lithium
Figure BDA0004113777320002171
(3R) -4- (2- ((4- (3-amino-6- (2- ((4- ((S) -6- (dimethylamino) -2- (1- (ethoxycarbonyl) cyclobutane-1-carboxamide) hexanamido) benzyl) oxy) phenyl) pyridazin-4-yl) -3, 8-diazabicyclo [3.2.1]Octan-8-yl) pyridin-2-yl) oxy ethyl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester (210 mg,0.200 mmol) and LiOH. H 2 A solution of O (25.6 mg,0.610 mmol) in tetrahydrofuran (3 mL) and water (1 mL) was stirred at room temperature for 1 hour. The solvent was concentrated under vacuum to give 254mg (crude) of the title compound as a yellow solid.
Step 3:1- (((2S) -1- ((4- ((2- (6-amino-5- (8- (2- (2- ((R) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) methyl) phenyl) amino) -6- (dimethylamino) -1-oxohex-2-yl) carbamoyl) cyclobutane-1-carboxylic acid
Figure BDA0004113777320002172
Will contain 1- (((2S) -1- ((4- ((2- (6-amino-5- (8- (2- ((R) -4- (tert-butoxycarbonyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]An octane-3-yl) pyridazin-3-yl-phenoxy) methyl) phenyl) -amino) -6- (dimethylamino) -1-oxohex-2-yl-carbamoyl) cyclobutane-1-carboxylic acid lithium (254 mg,0.250 mmol) in 5% TFA/HFIP (20 mL) was stirred at room temperature for 3 hours. The solvent was concentrated under vacuum. The residue was purified using a pre-packed C18 column (solvent gradient: 0-100% MeOH in water (0.05% NH) 4 HCO 3 ) The residue was purified to give 102mg (yield 44%) of the title compound as a red solid. LCMS (ESI) [ M+H ]] + ＝905。
Step 4:1- (((2S) -1- ((2- (6-amino-5- (8- (2- (2- ((R) -4- (2- ((5- ((R) -1- ((2S, 4R) -4-hydroxy-2- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) isoxazol-3-yl) oxy) ethyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) methyl) phenyl) amino) -6- (dimethylamino) -1-oxohex-2-yl) carbamoyl) cyclobutane-1-carboxylic acid
Figure BDA0004113777320002181
Will contain 1- (((2S) -1- ((4- ((2- (6-amino-5- (8- (2- ((R) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [ 3.2.1)]Octane-3-yl) pyridazin-3-yl) phenoxy-methyl) phenyl) -6- (dimethylamino) -1-oxohex-2-yl) carbamoyl) cyclobutane-1-carboxylic acid (102 mg,0.110 mmol), (2S, 4R) -4-hydroxy-1- ((R) -3-methyl-2- (3- (2-oxoethoxy) isoxazol-5-yl) butyryl) -N- ((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) pyrrolidine-2-carboxamide (61.0 mg,0.110 mmol) and CH3COOH (13.6 mg,0.230 mmol) in methanol (1.2 mL) and dichloromethane (0.4 mL) were stirred at room temperature for 1 hour. Then NaBH is added 3 CN (21.3 mg,0.340 mmol) was added thereto, and the mixture was stirred at room temperature for 0.5 hours. Water was added to quench the reaction. The solvent was concentrated under vacuum. The residue was purified using a pre-packed C18 column (solvent gradient: 0-100% MeOH in water (0.05% NH) 4 HCO 3 ) 80.0mg (49% yield) of the title compound was obtained as a yellow solid. LCMS (ESI) [ M+H ]] + ＝1429。
Step 4: n- ((2S) -1- ((4- ((2- (6-amino-5- (8- (2- (2- ((R) -4- (2- ((5- ((R) -1- ((2S, 4R) -4-hydroxy-2- (((S) -1- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) oxy) isoxazol-3-yl) oxy) ethyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) pyridazin-3-yl) phenoxy) methyl) amino) -6- (dimethylamino) -1-oxohex-2-yl) -N- (5- (2, 5-dioxo) -2, 5-dihydro-1H-pyrrol-1-yl) pentyl) cyclobutane-1, 1-dicarboxamide (2, 2-trifluoroacetate
Figure BDA0004113777320002191
To a solid phase containing 1- (((2S) -1- ((2- (6-amino-5- (8- (2- ((R) -4- (2- ((R) -1- ((2S, 4R) -4-hydroxy-2- (((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) carbamoyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) isoxazol-3-yl) oxy) ethyl) -2-methylpiperazin-1-yl) ethoxy) pyridin-4-yl) -3, 8-diazabicyclo [3.2.1 ] e at room temperature]To a solution of octane-3-yl) pyridazin-3-yl-phenoxy-methyl) phenyl) -amino) -6- (dimethylamino) -1-oxohex-2-yl-carbamoyl) cyclobutane-1-carboxylic acid (60.0 mg,0.0400 mmol), 1- (5-aminopentyl) -1H-pyrrole-2, 5-dione (2, 2-trifluoroacetic acid) (23.0 mg, crude product) and DIPEA (163 mg,1.26 mmol) in DMF (2 mL) was added HATU (24.0 mg,0.0600 mmol). The reaction was stirred at room temperature for 0.5 hours. By preparative HPLC (column: XBridge Prep Phenyl OBD column, 19X 250mm,5 μm; mobile phase A: water (0.05% FA), mobile phase B: ACN; flow rate: 25mL/min; gradient: increase from 17% B to 25% B,25% B over 10 min; wavelength: 254nm R T1 (min) 8.77). It was then passed through preparative HPLC (column: xselect CSH C18 OBD column 30X 150mm 5 μm; mobile phase A: water (0.05% TFA), mobile phase B: ACN; flow rate: 60mL/min; gradient: increase from 13% B to 38% B,38% B; wavelength: 254/220nm; RT in 8 minutes) 1 (min 8) purification to give 5.0mg (yield 7%) of L1-CIDE-BRM1-21 as a yellow solid. LCMS (ESI) [ M+H ]] + ＝1593。 1 H NMR(300MHz,DMSO-d6)δ10.21(s,1H),9.53(s,1H),8.99(s,1H),8.39(d,J＝7.8Hz,1H),7.95-7.80(m,3H),7.67(d,J＝8.1Hz,2H),7.63-7.50(m,2H),7.50-7.43(m,8H),7.19–7.08(m,1H),7.10-6.90(m,3H),6.64(s,1H),6.25(s,1H),6.11(s,1H),5.09(s,2H),4.91(t,J＝7.1Hz,1H),4.48(br,4H),4.41–4.30(m,4H),3.73-3.65(m,6H),3.47–3.31(m,7H),3.13–2.88(m,12H),2.75(d,J＝4.4Hz,7H),2.48-2.38(m,8H),2.25-2.15(m,1H),2.05–1.59(m,11H),1.50–1.29(m,8H),1.31-1.11(m,6H),0.96(d,J＝6.4Hz,3H),0.87–0.76(m,3H)。
Synthesis example 22
Conjugation of L1-CIDE to antibodies
Cysteine-engineered antibodies (THIOMAB) with 1M Tris TM ) The pH of 10mM succinate, pH 5, 150mM NaCl,2mM EDTA was adjusted to pH 7.5-8.5. 3-16 equivalents of L1-CIDE (containing thiol-reactive maleimide groups) were dissolved in DMF or DMA (concentration=10 mM) and added to the reduced, reoxidized and pH adjusted antibodies. The reaction was incubated at room temperature or 37 ℃ and monitored until the reaction was complete (1 to about 24 hours), as determined by LC-MS analysis of the reaction mixture. After completion of the reaction, ab-CIDE can be purified by one or any combination of several methods in order to remove residual unreacted linker drug intermediate and aggregated protein (if present at higher levels). In one example, ab-CIDE is diluted with 10mM histidine acetate (pH 5.5) until a final pH of about 5.5, and then purified by S cation exchange chromatography using a HiTrap S column, or S maxi centrifugal column (Pierce), connected to an Akta purification system (GE Healthcare). Alternatively, ab-CIDE is purified by gel filtration chromatography using an S200 column or a Zeba centrifugation column connected to an Akta purification system. Dialysis was used to purify the conjugate.
Thiomab using gel filtration or dialysis TM Ab-CIDE was formulated into 20mM His/acetate (pH 5) containing 240mM sucrose. Purified Ab-CIDE was concentrated by centrifugal ultrafiltration and filtered through a 0.2 μm filter under sterile conditions and stored frozen at-20 ℃.
Biological example 1:
cell level assay
Immunofluorescence detection of BRMs
Figure BDA0004113777320002201
Conjugation to CD22 had a DAR of 5.8. Conjugation to EpCAM had a DAR of 5.9.
Figure BDA0004113777320002211
Conjugation to CD22 had a DAR of 5.8. Conjugation to EpCAM had a DAR of 5.9. CD-22: thio Hu anti-CD 22F 4v3 high DAR [ LC: K149C HC: Y373CHC: L174C ] MeMe disulfide BRM CIDE; epCAM: thio Hu anti-Her 2 7C2 high DAR [ LC: K149C HC: L174C HC: Y373C ] MeMeMe disulfide BRM CIDE
FIGS. 1a and 1b show Ab-L1a-CIDE-BRM1-1 activity. FIGS. 2a and 2b show Ab-L1a-CIDE-BRM1-3 activity.
Biological example 2:
PK/PD BJAB tumor assay
The PK/PD effect of anti-CD 22-BRM Ab-CIDE was evaluated in a mouse xenograft model of BJAB-luc human non-Hodgkin's lymphoma. BJAB-luc was obtained from the Genntech cell line pool. The cell line was validated using a Short Tandem Repeat (STR) analysis of the Promega PowerPlex system and compared to the external STR profile of the cell line to confirm the family of the cell line.
To establish the model, tumor cells (2000 thousands in 0.2mL Hank balanced salt solution) were inoculated subcutaneously into the flank of female C.B-17SCID mice (Charles River Laboratories). When the tumors reached the required volume (300-400 mm 3), mice were randomly divided into n=5 groups, each group having a similar tumor size distribution, and received a single intravenous injection of vehicle (histidine buffer) or test substance via the tail vein. All anti-CD 22-BRM Ab-CIDE and unconjugated antibodies are formulated in histidine buffer (20 mM histidine acetate pH 5.5, 240mM sucrose, 0.02% Tween 20). Unconjugated BRM CIDE was formulated in 10% hydroxypropyl-beta-cyclodextrin, 50mM sodium acetate, pH 4.
Four days after dosing, mice were euthanized and tumors and whole blood collected. Tumors were resected and split into two equal parts, then flash frozen in liquid nitrogen. One was used to measure the level of BRM CIDE released and the other was used to assess the modulation of downstream PD markers. Whole blood was collected by terminal cardiac puncture under anesthesia surgery and placed in a test tube containing lithium heparin. The blood sample was placed on wet ice until centrifugation (within 15 minutes after collection). The samples were centrifuged at 10,000rpm for 5 minutes at 4 ℃ and plasma was collected, placed on dry ice and stored at-70 ℃ until analysis of linker stability and total antibody pharmacokinetics.
Western blot of xenograft tissues
Frozen tissue was cut into pieces of 15-30mg on dry ice and then transferred to 1.5mL Eppendorf safety lock tube with a 3.2mm (nexthavance (3.2 mm, ssb 32)) stainless steel ball. RIPA buffer (350 uL) (supplemented with 0.5M NaCl and freshly added 1 xhat protease and phosphatase inhibitors) was added and the sample tubes were placed into a bullets Blender tissue homogenizer. The sample was homogenized at the highest speed for 3 minutes. The sample tubes were centrifuged at maximum speed in a bench top centrifuge at 4 ℃ for 5 minutes and the lysate was transferred to new sample tubes. Protein concentration was determined using Pierce BCA protein assay. Protein lysates were prepared with sample buffer and reducing agent and incubated at 95℃for 3 min. Proteins (12 ug) were separated on 3-8% Tris acetate gel with Tris-acetate running buffer and then transferred onto nitrocellulose membrane using an iBlot transfer device (25V, 10 min). After blocking the membrane with TBS-T containing 5% milk for 30 minutes, primary antibody was added at 1/1000. Membranes were blotted against SMARCA2 (BRM) (rabbit, cell signaling technologies catalog No. 11966) and HDAC1 (mouse, cell signaling technologies catalog No. 5356) and incubated overnight on a rocker at 4 ℃. The next day, the membranes were washed with TBS-T on a shaker for 30 minutes at room temperature and the wash buffer was changed at least 3 times. The membrane was then incubated with 1/5000 Licor seconds in TBS-T for 1 hour at room temperature on a shaker. The blots were washed with TBS-T for 1 hour and the wash buffer was changed at least 6 times. Images were captured on a Licor imaging system.
Murine tumor assays were performed. Table 1 shows study groups and parameters.
TABLE 1 BJAB tumor (CB 17-SCID mouse), PK/PD study
Figure BDA0004113777320002221
Dividing tumor tissue into 2 parts for (1) BRM, BRG, PBRM PD and (2) tumor PK, respectively
Plasma PK time points were identical to the PD time points listed above
Antibody conjugate, IV formulation: histidine buffer, dose volume = 5mL/kg
CIDE-BRM1-3, IV formulation: 10% HP-b-CD and 50mM sodium acetate in water (pH 4.0), dosage volume = 5mL/kg
The dosage and antigen-dependent antitumor activity of Ab-L1a-CIDE-BRM1-1 are shown in FIGS. 3A-3L, and the dosage and antigen-dependent antitumor activity of Ab-L1a-CIDE-BRM1-3 are shown in FIGS. 4A-4L.
Biological example 3:
target protein degradation assay
Data reporting on improved PD response. The data shown in FIG. 5 indicate that Ab-L1a-CIDE-BRM1-1, BRM and BRG1 degradation is associated with antitumor activity. The data shown in FIG. 6 demonstrate that Ab-L1a-CIDE-BRM1-3, BRM and BRG1 degradation has a low correlation with antitumor activity. The data shown in fig. 7 demonstrate that the antibody conjugation strategy increases degradation activity. The time points at which these data were obtained were all 96 hours. Ab-L1a-CIDE-BRM1-1 degrades to a greater extent than unconjugated CIDE-BRM1-3, whereas both compounds have similar BRM degradation characteristics in unconjugated forms of the cellular assay (CIDE-BRM 1-3 and CIDE-BRM1-1 assay described in WO 2019195201). This effect suggests that the ligation strategy described herein may modulate degradation characteristics.
Biological example 4:
cell assay for determining DC50 and Dmax
Cell level assays were performed in both cell lines to determine DC50 and Dmax for Ab-L1-CIDE. BJAB, HCC515 and H1944 cells were seeded in 384-well plates at densities of 5000, 4000 and 2500 cells/well, respectively. The next day, ab-CIDE was added. After 24 hours of drug treatment, the cells were fixed with 4% formaldehyde for 15 minutes. The well plate was washed three times with PBS. Cells were incubated with IF blocking solution (10% FCS, 1% BSA, 0.1% Triton, 0.01% azide, X-100 in PBS). After 1.5 hours, add primary antibody solution diluted 2X in IF blocking buffer: BRM is added (Cell signaling catalog number 11966, 1:2000). The well plate was incubated overnight at 4 ℃. The next morning, wash with PBSCells were three times. Cells were then incubated with secondary antibody (rabbit-Alexa 488A21206 (1:2000)) at room temperature in the dark for 1 hour. Hoechst H3570 was added to the wells at 1:5000 and the well plates were incubated for an additional 30 minutes. The well plate was washed with 3xPBS and dried in Opera Phenix TM Imaging on a high content screening system. The average signal intensity of nuclear BRMs was quantified using nuclear staining as a mask.
The data are shown in table 2 below. The data demonstrate the success of the antibody targeting strategy disclosed herein. Negative control: anti-gD and anti-TROP 2 do not interact with NCI-H1944 cells, while anti-TfR 2 interacts with the cells. anti-gD further did not interact with HCC515 cells, while anti-TfR 2 and anti-TROP 2 interacted with the cells. The data show that several Ab-L1-CIDE have the required low DC50 and the required high Dmax values.
Table 2.
Figure BDA0004113777320002241
Biological example 5:
lysosomal release assay
Lysosomal release assays were run to measure the release of degradants from the L1 moiety in an environment that mimics the intracellular environment. In order for the degradant to be active in binding to BRM and transporting it to ubiquitin ligase, L1 must first be released.
The assay was run using L1 bound to BRM binding compound, designated "L1-BRM1- #", corresponding to the respective CIDE. This test determines whether cleavage of the covalent linkage of L1 to the BRM moiety occurs. The linker drug (10. Mu.M) was incubated with human liver lysosomes (0.17 mg/mL) and cysteine (5 mM) in 100mM citrate buffer (pH 5.5) for 24 hours. The samples were analyzed by Q Exactive Orbitrap mass spectrometer, wherein the gradient elution was performed using an LC mobile phase containing (a) 0.1% aqueous formic acid solution and (B) 0.1% acetonitrile formic acid solution.
The measurement results are shown in table 3 below. The results indicate that the direct linking strategy of L1 with BRM moieties is released in the cellular environment. The conjugate L1-CIDE-BRM1-15 does not comprise an antibody linker of the linker-1 type described herein. In the DAC tested, L1-CIDE-BRM1-15 did not release the degradant in the lysosomal extract. This finding supports strategies for selectively attaching degradants to abs, such as the L1 linker of the linker 1 type described herein.
Table 3.
Figure BDA0004113777320002251
* "No" means that <15% free drug was observed after 24 hours incubation in lysosomal extracts at 37 ℃. "yes" means that >50% free drug is observed after 24 hours incubation in lysosomal extracts at 37 ℃.
Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for.
Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used in the practice of the subject matter described herein. The present invention is in no way limited to the methods and materials described.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this subject matter belongs and are consistent with the following documents: singleton et al (1994) Dictionary of Microbiology and Molecular Biology, 2 nd edition, j.wiley & Sons, new York, NY; and Janeway, c., convers, p., walport, m., shomchik (2001) Immunobiology, 5 th edition, garland Publishing, new York.
In the description and claims, the words "comprising," "including," and "containing" are used in a non-exclusive sense unless the context requires otherwise. It is to be understood that the embodiments described herein include "consisting of" and/or "consisting essentially of" the embodiments.
The term "about," as used herein, when referring to a numerical value, means to include a variation from a specified amount, including in some embodiments ±50% of the specified amount, including in some embodiments ±20% of the specified amount, including in some embodiments ±10% of the specified amount, including in some embodiments ±5% of the specified amount, including in some embodiments ±1% of the specified amount, including in some embodiments ±0.5% of the specified amount, including in some embodiments ±0.1% of the specified amount, as such variation is suitable for performing the disclosed methods or employing the disclosed compositions.
If a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the same reference. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.
Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
Figure IDA0004113777440000011
Figure IDA0004113777440000021
Figure IDA0004113777440000031
Figure IDA0004113777440000041
Figure IDA0004113777440000051
Figure IDA0004113777440000061
Figure IDA0004113777440000071
Figure IDA0004113777440000081
Figure IDA0004113777440000091
Figure IDA0004113777440000101
Figure IDA0004113777440000111
Figure IDA0004113777440000121
Figure IDA0004113777440000131
Figure IDA0004113777440000141
Figure IDA0004113777440000151
Figure IDA0004113777440000161
Figure IDA0004113777440000171
Figure IDA0004113777440000181
Figure IDA0004113777440000191
Figure IDA0004113777440000201
Figure IDA0004113777440000211
Figure IDA0004113777440000221
Figure IDA0004113777440000231
Figure IDA0004113777440000241
Figure IDA0004113777440000251
Figure IDA0004113777440000261
Figure IDA0004113777440000271
Figure IDA0004113777440000281
Figure IDA0004113777440000291
Figure IDA0004113777440000301
Figure IDA0004113777440000311

Claims (72)

1. A conjugate having the structure:
Ab-(L1-D) p ，
wherein ,
ab is an antibody;
d is CIDE or prodrug thereof, having the following structure:
Figure FDA0004113777300000011
wherein ,
BRM is the residue of a BRM binding compound,
e3LB is the residue of an E3 ligase binding compound, and
l2 is a moiety that covalently links BRM and E3 LB;
l1 is linker-1 covalently linking Ab to one of BRM, E3LB, or L2; and is also provided with
p is 1 to 16.
2. The conjugate of claim 1, wherein L1 is covalently bound to E3 LB.
3. The conjugate of claim 2, wherein the prodrug is a CIDE having a phosphate moiety covalently bound to BRM.
4. The conjugate of claim 1, wherein L1 is covalently bound to BRM.
5. The conjugate of claim 4, wherein the prodrug is CIDE having a phosphate moiety covalently bound to E3 LB.
6. The conjugate of any one of claims 3 or 5, wherein the phosphate moiety has the structure:
Figure FDA0004113777300000012
wherein e is 0 or 1.
7. The conjugate of claim 1, wherein L1 is covalently bound to L2.
8. The conjugate of claim 1, wherein L1 is selected from the group consisting of:
i)L1a
Figure FDA0004113777300000021
wherein
wherein Ra 、R b 、R c and Rd Independently selected from H, optionally substituted branched or straight chain C 1 -C 5 Alkyl and optionally substituted C 3 -C 6 Cycloalkyl group, or R a and Rb Or R is c and Rd Together with the carbon atoms to which they are bound form an optionally substituted C 3 -C 6 Cycloalkyl ring or 3-to 6-membered heterocycloalkyl ring;
ii)L1b
Figure FDA0004113777300000022
wherein ,
Z and Z1 Each independently is C 1-12 Alkylene or- - [ CH ] 2 ] g -[-O-CH 2 ] h -, wherein g is 0, 1 or 2, and h is 1 to 5;
R z is H or C 1-3 An alkyl group; and
iii)L1c
Figure FDA0004113777300000023
wherein
Z 2 Is C 1-12 Alkylene or- [ CH ] 2 ] g -[-O-CH 2 ] h -, wherein g is 0, 1 or 2, and h is 1 to 5;
w is 1, 2, 3, 4 or 5;
j is-N (R) x )(R y )、-C(O)NH 2 、-NH-C(O)-NH 2 、-NH-NH-NH 2, wherein ,Rx and Ry Each independently selected from hydrogen and C 1-3 An alkyl group;
k is selected from-CH 2 -、-CH(R)-、-CH(R)-O-^、-C(O)-、^-C(O)-O-CH(R)-、-CH 2 -O-C(O)-^、-CH 2 -O-C(O)-NH-^、^-O-C(L1c)-C(O)-NR x R y -、^-C(L1c)-C(O)-NR x R y -、-CH 2 -O-C(O)-NH-CH 2 -、-CH 2 -O-C(O)-R-[CH 2 ] q -O-^、-CH 2 -O-C(O)-R-[CH 2 ] q -the process comprises, where a represents the connection to CIDE, wherein R is hydrogen, C 1-3 Alkyl, N (R) x )(R y )、-O-N(R x )(R y ) Or C (O) -N (R) x )(R y ) Wherein q is 0, 1, 2 or 3, and R x and Ry Each independently selected from hydrogen and C 1-3 Alkyl, or R x and Ry Together with the nitrogen to which each is attached, form an optionally substituted 5-to 7-membered heterocyclyl;
ra and Rb are each independently selected from hydrogen and C 1-3 Alkyl, or Ra and Rb, together with the nitrogen to which each is attached, form an optionally substituted C 3-6 Cycloalkyl; and is also provided with
R 7 and R8 Each independently is hydrogen, halo, C 1-5 Alkyl, C 1-5 Alkoxy or hydroxy.
9. The conjugate of claim 8, wherein L1a is covalently bound to E3 LB.
10. The conjugate of claim 8, wherein L1b is covalently bound to E3LB or BRM.
11. The conjugate of claim 8, wherein L1c is covalently bound to E3LB, BRM or L2.
12. The conjugate of claim 8, wherein L1b is covalently bound to E3 LB.
13. The conjugate of claim 8, wherein L1b is covalently bound to BRM.
14. The conjugate of claim 8, wherein L1c is covalently bound to E3 LB.
15. The conjugate of claim 8, wherein L1c is covalently bound to BRM.
16. The conjugate of claim 8, wherein L1c is covalently bound to L2.
17. The conjugate of claim 1, wherein D has the structure:
Figure FDA0004113777300000031
wherein L1 is attached at one attachment point selected from the group consisting of: L1-Q, L1-Q', L1-S, L1-T and optionally L1-U, L1-V and L1-Y, if present, wherein
L1Q on BRM
Figure FDA0004113777300000032
Where M is O;
L1-Q' on BRM
Figure FDA0004113777300000041
Where M' is-NH;
L1-S on L2
Figure FDA0004113777300000042
Figure FDA0004113777300000043
A place;
L1-T on E3LB
Figure FDA0004113777300000044
Wherein A is a group covalently bonded to L2;
L1-U and L1-V on E3LB
Figure FDA0004113777300000045
A place; and is also provided with
L1-Y on E3LB
Figure FDA0004113777300000046
Figure FDA0004113777300000051
At the position of the first part,
wherein ,
Figure FDA0004113777300000052
is a single bond or a double bond.
18. The conjugate of claim 8, wherein K of L1c is selected from the group consisting of:
L1c-CH2-、
Figure FDA0004113777300000053
Figure FDA0004113777300000054
19. the conjugate of claim 17, having the structure:
Figure FDA0004113777300000061
wherein ：
R 3 is cyano group,
Figure FDA0004113777300000062
Figure FDA0004113777300000063
wherein ,
Figure FDA0004113777300000064
is a single bond or a double bond.
20. The conjugate of claim 19, having the structure:
Figure FDA0004113777300000065
wherein ,R1A 、R 1B and R1C Each independently is hydrogen or C 1-5 An alkyl group; or alternatively
R 1A 、R 1B and R1C Two of (3)Which together with the carbon to which each is attached form C 1-5 Cycloalkyl groups.
21. The conjugate of claim 20, wherein R 1A 、R 1B and R1C Each independently is hydrogen or methyl.
22. The conjugate of claim 21, wherein R 1A and R1B Each methyl.
23. The conjugate of claim 22, having the structure:
Figure FDA0004113777300000071
/>
24. the conjugate of claim 23, wherein R 2 Is hydrogen, methyl, ethyl or propyl.
25. The conjugate of claim 24, wherein R 2 Is methyl.
26. The conjugate of claim 25, wherein R 2 Combined with E3LB to
Figure FDA0004113777300000081
27. The conjugate of claim 23, wherein Y 1 and Y2 Each is-CH.
28. The conjugate of claim 23, wherein Y 1 Is N, and Y 2 is-CH.
29. The conjugate of claim 23, wherein Y 1 is-CH, and Y 2 Is N.
30. The conjugate of claim 23, wherein L1 is attached at L1-Q, L1-Q' or L1-T.
31. The conjugate of claim 30, having the structure:
Figure FDA0004113777300000082
32. the conjugate of claim 17, wherein:
l1 is attached at L1-T and is
Figure FDA0004113777300000091
Wherein Ra, rb, rc and Rd are each independently selected from hydrogen and C 1-3 An alkyl group.
33. The conjugate of claim 17, wherein:
l1 is attached at L1-T and is
Figure FDA0004113777300000092
/>
Wherein Ra, rb, rc and Rd are each independently selected from hydrogen and C 1-3 An alkyl group; and is also provided with
A phosphate moiety having the structure:
Figure FDA0004113777300000093
Wherein e is 0 or 1,
covalently binds to BRM.
34. The conjugate of claim 17, wherein:
l1 is attached at L1-T and is selected from the group consisting of:
ii)L1b
Figure FDA0004113777300000094
and
iii)L1c
Figure FDA0004113777300000101
35. the conjugate of claim 17, wherein:
l1 is attached at L1-T and is selected from the group consisting of:
ii)L1b
Figure FDA0004113777300000102
and
iii)L1c
Figure FDA0004113777300000103
and a phosphate moiety having the structure: />
Figure FDA0004113777300000104
Wherein e is 0 or 1,
covalently binds to BRM.
36. The conjugate of claim 17, wherein:
l1 is attached at L1-Q and is selected from the group consisting of:
ii)L1b
Figure FDA0004113777300000111
and
iii)L1c
Figure FDA0004113777300000112
37. the conjugate of claim 17, wherein:
l1 is attached at L1-Q and is selected from the group consisting of:
ii)L1b
Figure FDA0004113777300000113
and
iii)L1c
Figure FDA0004113777300000121
and
A phosphate moiety having the structure:
Figure FDA0004113777300000122
wherein e is 0 or 1,
covalently binds to BRM.
38. The conjugate of claim 17, wherein:
l1 is attached at L1-Q' and has the structure:
iii)L1c
Figure FDA0004113777300000123
39. the conjugate of claim 17, wherein:
l1 is attached at L1-Q' and has the structure:
iii)L1c
Figure FDA0004113777300000124
and
A phosphate moiety having the structure:
Figure FDA0004113777300000131
wherein e is 0 or 1,
covalently binds to E3 LB.
40. The conjugate of claim 8, wherein:
Z and Z1 Each independently selected from- (CH) 2 ) 1-6- and -[CH2 ] g -[-O-CH 2 ] h -, wherein g is 0, 1 or 2, and h is 1 to 5.
41. The conjugate of claim 8, wherein:
Z 2 selected from- (CH) 2 ) 1-6- and -[CH2 ] g -[-O-CH 2 ] h -, wherein g is 0, 1 or 2, and h is 1 to 5.
42. The conjugate of claim 8, wherein L1 is selected from the group consisting of:
L1a-i)
Figure FDA0004113777300000132
L1a-ii)
Figure FDA0004113777300000133
L1a-iii)
Figure FDA0004113777300000134
L1b-i)
Figure FDA0004113777300000141
L1c-i)
Figure FDA0004113777300000142
L1c-ii)
Figure FDA0004113777300000143
L1c-iii)
Figure FDA0004113777300000151
wherein ,
j is-CH 2 -CH 2 -CH 2 -NH-C(O)-NH 2 ；-CH 2 -CH 2 -CH 2 -CH 2 -NH 2 ；-CH 2 -CH 2 -CH 2 -CH 2 -NH-CH 3 The method comprises the steps of carrying out a first treatment on the surface of the or-CH 2 -CH 2 -CH 2 -CH 2 -
N(CH 3 ) 2 ；
R 5 and R6 Independently hydrogen or C 1-5 An alkyl group; or R is 5 and R6 Together with the nitrogen to which each is attached, form an optionally substituted 5-to 7-membered heterocyclyl; and is also provided with
R 7 and R8 Each independently is hydrogen, halo, C 1-5 Alkyl, C 1-5 Alkoxy or hydroxy.
43. The conjugate according to claim 42, wherein L1 is selected from the group consisting of:
Figure FDA0004113777300000152
wherein ,
j is-CH 2 -CH 2 -CH 2 -NH-C(O)-NH 2 ；-CH 2 -CH 2 -CH 2 -CH 2 -NH 2 ；-CH 2 -CH 2 -CH 2 -CH 2 -NH-CH 3 The method comprises the steps of carrying out a first treatment on the surface of the or-CH 2 -CH 2 -CH 2 -CH 2 -
N(CH 3 ) 2； and
R 7 and R8 Each independently is hydrogen, halo, C 1-5 Alkyl, C 1-5 Alkoxy or hydroxy.
44. The conjugate according to claim 43, wherein J is-CH 2 -CH 2 -CH 2 -NH-C(O)-NH 2 or-CH 2 -CH 2 -CH 2 -CH 2 -N(CH 3 ) 2 。
45. The conjugate according to claim 43, wherein L1 has the structure:
Figure FDA0004113777300000161
wherein ,
j is-CH 2 -CH 2 -CH 2 -NH-C(O)-NH 2 ；-CH 2 -CH 2 -CH 2 -CH 2 -NH 2 ；-CH 2 -CH 2 -CH 2 -CH 2 -NH-CH 3 The method comprises the steps of carrying out a first treatment on the surface of the or-CH 2 -CH 2 -CH 2 -CH 2 -N(CH 3 ) 2； and
R 7 and R8 Each independently is hydrogen, halo, C 1-5 Alkyl, C 1-5 Alkoxy or hydroxy.
46. The conjugate according to claim 45, wherein L1 has the structure:
Figure FDA0004113777300000162
47. The conjugate according to claim 43, wherein linker-1 has the structure:
Figure FDA0004113777300000171
wherein ,
j is-CH 2 -CH 2 -CH 2 -NH-C(O)-NH 2 ；-CH 2 -CH 2 -CH 2 -CH 2 -NH 2 ；-CH 2 -CH 2 -CH 2 -CH 2 -NH-CH 3 The method comprises the steps of carrying out a first treatment on the surface of the or-CH 2 -CH 2 -CH 2 -CH 2 -
N(CH 3 ) 2； and
R 7 and R8 Each independently is hydrogen, halo, C 1-5 Alkyl, C 1-5 Alkoxy or hydroxy.
48. The conjugate according to claim 47, wherein L1 has the structure:
Figure FDA0004113777300000172
49. the conjugate of claim 1, wherein:
Figure FDA0004113777300000173
residues of BRM binding compounds having the structure of formula I:
Figure FDA0004113777300000181
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any of the foregoing, wherein: wherein X is hydrogen or halogen;
Figure FDA0004113777300000182
selected from the group consisting of:
(a)
Figure FDA0004113777300000183
(b)
Figure FDA0004113777300000184
/>
(c)
Figure FDA0004113777300000185
(d)
Figure FDA0004113777300000186
and
(e)
Figure FDA0004113777300000191
wherein for (a) to (e), represents the point of attachment to [ X ] or, if [ X ] is absent, the point of attachment to [ Y ] and the point of attachment to the phenyl ring; and wherein:
(i)[X]is a 3-to 15-membered heterocyclyl or a 5-to 20-membered heteroaryl, provided that when
Figure FDA0004113777300000192
In the case of (a), then [ X ]]Not be
Figure FDA0004113777300000193
Figure FDA0004113777300000194
Wherein # represents->
Figure FDA0004113777300000195
And # denotes the connection point with L2,
[ Y ] is absent, and
[ Z ] is absent; or alternatively
(ii)[X]Is a 3-to 15-membered heterocyclyl or a 5-to 20-membered heteroaryl, wherein [ X ]]Optionally substituted 3-to 15-membered heterocyclyl with one or more-OH or C 1-6 An alkyl group is substituted and a substituent is substituted,
[ Y ] is absent, and
[ Z ] is a 3-to 15-membered heterocyclic group or a 5-to 20-membered heteroaryl group,
provided that when
Figure FDA0004113777300000196
Is (a) and [ X ]]Is->
Figure FDA0004113777300000197
In which&Representation and->
Figure FDA0004113777300000198
And (2) connection point of&&Representation and [ Z ]]And [ Z ]]Not->
Figure FDA0004113777300000199
Wherein # denotes an integer of and [ X ]]And # denotes the connection point to L2; or alternatively
(iii) [ X ] is a 3-to 15-membered heterocyclic group or a 5-to 20-membered heteroaryl group,
[ Y ] is a methylene group, wherein the methylene group of [ Y ] is optionally substituted with one or more methyl groups, and
[ Z ] is a 3-to 15-membered heterocyclic group; or alternatively
(iv) [ X ] is not present and,
[ Y ] is a vinylidene group, wherein the vinylidene group of [ Y ] is optionally substituted with one or more halo groups, and
[ Z ] is a 5-to 20-membered heteroaryl,
provided that the conditions are that,
Figure FDA0004113777300000201
is (a), (b), (d) or (e); or alternatively
(v) [ X ] is not present and,
[ Y ] is ethynylene, and
[ Z ] is a 5-to 20-membered heteroaryl,
provided that the conditions are that,
Figure FDA0004113777300000202
is (a), (b), (d) or (e); or alternatively
(vi) [ X ] is not present and,
[ Y ] is cyclopropyl or cyclobutyl, and
[ Z ] is a 5-to 20-membered heteroaryl,
provided that the conditions are that,
Figure FDA0004113777300000203
is (a), (b), (d) or (e).
50. The conjugate according to claim 49, wherein the residue of BRM binding compound is a compound of formula (I-a):
Figure FDA0004113777300000204
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt thereof.
51. The conjugate according to claim 49, wherein the residue of BRM binding compound is a compound of formula (I-B):
Figure FDA0004113777300000211
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt thereof.
52. The conjugate according to claim 49, wherein the residue of a BRM binding compound is a compound of formula (I-C):
Figure FDA0004113777300000212
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt thereof.
53. The conjugate according to claim 49, wherein the residue of BRM binding compound is a compound of formula (I-D):
Figure FDA0004113777300000213
wherein X is hydrogen or halogen,
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt thereof.
54. The conjugate according to claim 49, wherein the residue of BRM binding compound is a compound of formula (I-E):
Figure FDA0004113777300000221
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt thereof.
55. The conjugate of claim 49, wherein the residue of BRM binding compound comprises at least one moiety selected from the group consisting of:
Figure FDA0004113777300000222
wherein M is-NH or oxygen and is covalently bonded to L1-Q.
56. The conjugate of claim 55, wherein the residue of BRM binding compound comprises a moiety selected from the group consisting of:
Figure FDA0004113777300000223
57. The conjugate according to claim 56, wherein BRM is a residue of:
Figure FDA0004113777300000231
/>
wherein ,
Figure FDA0004113777300000241
is the connection point with L2.
58. The conjugate of claim 1, wherein:
l2 is a linker-2 covalently bound to E3LB and BRM, said L2 having the formula:
Figure FDA0004113777300000242
wherein ,
R 4 is hydrogen or methyl, and is preferably hydrogen or methyl,
Figure FDA0004113777300000243
wherein ,
z is 1 or 0 and is preferably selected from the group consisting of,
g is
Figure FDA0004113777300000244
or-C (O) NH-; and is also provided with
Figure FDA0004113777300000245
Is the point of attachment to the BRM.
59. The conjugate according to claim 58, wherein R 4 Is hydrogen.
60. The conjugate according to claim 58, wherein R 4 Is methyl.
61. The conjugate according to claim 60, wherein R 4 Methyl is shown below:
Figure FDA0004113777300000251
62. the conjugate of claim 1, selected from the group consisting of:
Figure FDA0004113777300000252
/>
Figure FDA0004113777300000261
/>
Figure FDA0004113777300000271
Figure FDA0004113777300000281
/>
63. the conjugate of claim 1, selected from the group consisting of:
Figure FDA0004113777300000282
Figure FDA0004113777300000291
64. the conjugate of claim 1, selected from the group consisting of:
Figure FDA0004113777300000292
Figure FDA0004113777300000301
65. the conjugate of claim 1, wherein p has a value of about 5 to about 14.
66. The conjugate of claim 1, wherein p has a value of about 5 to about 10.
67. A pharmaceutical composition comprising the conjugate of claim 1 and one or more pharmaceutically acceptable excipients.
68. A method of treating a disease in a human in need thereof, comprising administering to the human an effective amount of the conjugate of claim 1 or the composition of claim 47.
69. The method of claim 68, wherein the disease is cancer.
70. The method of claim 68, wherein the cancer is BRM-dependent.
71. The method of claim 68, wherein the cancer is non-small cell lung cancer.
72. A method of reducing target BRM protein levels in a subject, comprising:
administering to the subject the conjugate of claim 1 or the composition of claim 69, wherein the BRM moiety binds to the target BRM protein, wherein ubiquitin ligase effects degradation of the bound target BRM protein, wherein the level of the BRM target protein is reduced.
CN202180060539.4A 2020-07-21 2021-07-20 Antibody conjugation chemical inducer for degrading BRM and method thereof Pending CN116249556A (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US202063054757P 2020-07-21 2020-07-21
US63/054,757 2020-07-21
PCT/US2021/042280 WO2022020288A1 (en) 2020-07-21 2021-07-20 Antibody-conjugated chemical inducers of degradation of brm and methods thereof

Publications (1)

Publication Number Publication Date
CN116249556A true CN116249556A (en) 2023-06-09

Family

ID=77265314

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202180060539.4A Pending CN116249556A (en) 2020-07-21 2021-07-20 Antibody conjugation chemical inducer for degrading BRM and method thereof

Country Status (17)

Country Link
US (1) US20230330249A1 (en)
EP (1) EP4185328A1 (en)
JP (1) JP2023535409A (en)
KR (1) KR20230042032A (en)
CN (1) CN116249556A (en)
AR (1) AR123019A1 (en)
AU (1) AU2021312225A1 (en)
BR (1) BR112023001143A2 (en)
CA (1) CA3188649A1 (en)
CL (1) CL2023000193A1 (en)
CO (1) CO2023000679A2 (en)
CR (1) CR20230017A (en)
IL (1) IL299860A (en)
MX (1) MX2023000888A (en)
PE (1) PE20231104A1 (en)
TW (1) TW202216215A (en)
WO (1) WO2022020288A1 (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AR128331A1 (en) * 2022-01-26 2024-04-17 Genentech Inc CHEMICAL DEGRADATION INDUCTORS CONJUGATED WITH ANTIBODIES AND METHODS OF THESE
AR128330A1 (en) * 2022-01-26 2024-04-17 Genentech Inc CHEMICAL DEGRADATION INDUCERS CONJUGATED WITH ANTIBODY AND METHODS OF THESE
WO2023216009A1 (en) * 2022-05-07 2023-11-16 哈尔滨工业大学 Small molecule-nanoantibody conjugate inducer of proximity (snacip), and preparation method therefor and use thereof

Family Cites Families (76)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US5807715A (en) 1984-08-27 1998-09-15 The Board Of Trustees Of The Leland Stanford Junior University Methods and transformed mammalian lymphocyte cells for producing functional antigen-binding protein including chimeric immunoglobulin
US4676980A (en) 1985-09-23 1987-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target specific cross-linked heteroantibodies
US6548640B1 (en) 1986-03-27 2003-04-15 Btg International Limited Altered antibodies
ES2052027T5 (en) 1988-11-11 2005-04-16 Medical Research Council IMMUNOGLOBULINE VARIABLE DOMAIN SEQUENCE CLONING.
DE3920358A1 (en) 1989-06-22 1991-01-17 Behringwerke Ag BISPECIFIC AND OLIGO-SPECIFIC, MONO- AND OLIGOVALENT ANTI-BODY CONSTRUCTS, THEIR PRODUCTION AND USE
US5208020A (en) 1989-10-25 1993-05-04 Immunogen Inc. Cytotoxic agents comprising maytansinoids and their therapeutic use
US5959177A (en) 1989-10-27 1999-09-28 The Scripps Research Institute Transgenic plants expressing assembled secretory antibodies
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
US6075181A (en) 1990-01-12 2000-06-13 Abgenix, Inc. Human antibodies derived from immunized xenomice
US5770429A (en) 1990-08-29 1998-06-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
ES2113940T3 (en) 1990-12-03 1998-05-16 Genentech Inc ENRICHMENT METHOD FOR PROTEIN VARIANTS WITH ALTERED UNION PROPERTIES.
US5571894A (en) 1991-02-05 1996-11-05 Ciba-Geigy Corporation Recombinant antibodies specific for a growth factor receptor
JP4124480B2 (en) 1991-06-14 2008-07-23 ジェネンテック・インコーポレーテッド Immunoglobulin variants
GB9114948D0 (en) 1991-07-11 1991-08-28 Pfizer Ltd Process for preparing sertraline intermediates
JP3951062B2 (en) 1991-09-19 2007-08-01 ジェネンテック・インコーポレーテッド Expression of antibody fragments with cysteine present at least as a free thiol in E. coli for the production of bifunctional F (ab ') 2 antibodies
US5587458A (en) 1991-10-07 1996-12-24 Aronex Pharmaceuticals, Inc. Anti-erbB-2 antibodies, combinations thereof, and therapeutic and diagnostic uses thereof
WO1993008829A1 (en) 1991-11-04 1993-05-13 The Regents Of The University Of California Compositions that mediate killing of hiv-infected cells
WO1993016185A2 (en) 1992-02-06 1993-08-19 Creative Biomolecules, Inc. Biosynthetic binding protein for cancer marker
US6214345B1 (en) 1993-05-14 2001-04-10 Bristol-Myers Squibb Co. Lysosomal enzyme-cleavable antitumor drug conjugates
US5789199A (en) 1994-11-03 1998-08-04 Genentech, Inc. Process for bacterial production of polypeptides
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
US5840523A (en) 1995-03-01 1998-11-24 Genetech, Inc. Methods and compositions for secretion of heterologous polypeptides
US5869046A (en) 1995-04-14 1999-02-09 Genentech, Inc. Altered polypeptides with increased half-life
US6319688B1 (en) 1997-04-28 2001-11-20 Smithkline Beecham Corporation Polynucleotide encoding human sodium dependent phosphate transporter (IPT-1)
JP4213224B2 (en) 1997-05-02 2009-01-21 ジェネンテック，インコーポレーテッド Method for producing multispecific antibody having heteromultimer and common component
US6040498A (en) 1998-08-11 2000-03-21 North Caroline State University Genetically engineered duckweed
US6610833B1 (en) 1997-11-24 2003-08-26 The Institute For Human Genetics And Biochemistry Monoclonal human natural antibodies
EP1034298B1 (en) 1997-12-05 2011-11-02 The Scripps Research Institute Humanization of murine antibody
WO2001057188A2 (en) 2000-02-03 2001-08-09 Hyseq, Inc. Novel nucleic acids and polypeptides
US7125978B1 (en) 1999-10-04 2006-10-24 Medicago Inc. Promoter for regulating expression of foreign genes
CA2385347C (en) 1999-10-04 2009-12-15 Medicago Inc. Method for regulating transcription of foreign genes
AU784983B2 (en) 1999-12-15 2006-08-17 Genentech Inc. Shotgun scanning, a combinatorial method for mapping functional protein epitopes
AU2001253140A1 (en) 2000-04-03 2001-10-15 The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Tumor markers in ovarian cancer
IL151853A0 (en) 2000-04-11 2003-04-10 Genentech Inc Multivalent antibodies and uses therefor
US6596541B2 (en) 2000-10-31 2003-07-22 Regeneron Pharmaceuticals, Inc. Methods of modifying eukaryotic cells
CN1487996B (en) 2000-11-30 2010-06-16 米德列斯公司 Transgenic transchromosomal rodents for making human antibodies
MXPA03011979A (en) 2001-06-18 2005-04-08 Eos Biotechnology Inc Methods of diagnosis of ovarian cancer, compositions and methods of screening for modulators of ovarian cancer.
JP2005526044A (en) 2002-02-21 2005-09-02 デューク ユニバーシティ Treatment method using anti-CD22 antibody
US9770517B2 (en) 2002-03-01 2017-09-26 Immunomedics, Inc. Anti-Trop-2 antibody-drug conjugates and uses thereof
EP1513879B1 (en) 2002-06-03 2018-08-22 Genentech, Inc. Synthetic antibody phage libraries
JP2004121218A (en) 2002-08-06 2004-04-22 Jenokkusu Soyaku Kenkyusho:Kk Method for testing bronchial asthma or chronic obstructive pulmonary disease
AU2002951346A0 (en) 2002-09-05 2002-09-26 Garvan Institute Of Medical Research Diagnosis of ovarian cancer
AU2003288918A1 (en) 2002-10-04 2004-05-04 Van Andel Research Institute Molecular sub-classification of kidney tumors and the discovery of new diagnostic markers
CA2499300A1 (en) 2002-10-31 2004-05-21 Genentech, Inc. Methods and compositions for increasing antibody production
EP1585767A2 (en) 2003-01-16 2005-10-19 Genentech, Inc. Synthetic antibody phage libraries
WO2004065417A2 (en) 2003-01-23 2004-08-05 Genentech, Inc. Methods for producing humanized antibodies and improving yield of antibodies or antigen binding fragments in cell culture
KR101192496B1 (en) 2003-11-06 2012-10-18 시애틀 지네틱스, 인크. Monomethylvaline compounds capable of conjugation to ligands
EP1740615B1 (en) 2004-03-31 2014-11-05 Genentech, Inc. Humanized anti-tgf-beta antibodies
US7785903B2 (en) 2004-04-09 2010-08-31 Genentech, Inc. Variable domain library and uses
ES2577292T3 (en) 2005-11-07 2016-07-14 Genentech, Inc. Binding polypeptides with diversified VH / VL hypervariable sequences and consensus
US20070237764A1 (en) 2005-12-02 2007-10-11 Genentech, Inc. Binding polypeptides with restricted diversity sequences
CA2651567A1 (en) 2006-05-09 2007-11-22 Genentech, Inc. Binding polypeptides with optimized scaffolds
EP2035456A1 (en) 2006-06-22 2009-03-18 Novo Nordisk A/S Production of bispecific antibodies
US10118970B2 (en) 2006-08-30 2018-11-06 Genentech, Inc. Multispecific antibodies
CN100592373C (en) 2007-05-25 2010-02-24 群康科技(深圳)有限公司 Liquid crystal panel drive device and its drive method
PL2235064T3 (en) 2008-01-07 2016-06-30 Amgen Inc Method for making antibody fc-heterodimeric molecules using electrostatic steering effects
EP2303332B1 (en) 2008-07-15 2014-12-31 Genentech, Inc. Anthracycline conjugates, process for their preparation and their use as antitumor compounds
US9249145B2 (en) 2009-09-01 2016-02-02 Incyte Holdings Corporation Heterocyclic derivatives of pyrazol-4-yl-pyrrolo[2,3-d]pyrimidines as janus kinase inhibitors
CN105001330B (en) 2010-04-23 2020-05-01 弗·哈夫曼-拉罗切有限公司 Production of heteromultimeric proteins
AR085138A1 (en) 2011-02-04 2013-09-11 Genentech Inc Fc VARIATIONS AND METHODS FOR PRODUCTION
WO2013017705A1 (en) 2011-08-03 2013-02-07 Salvador Moreno Rufino Baltasar Panel system for construction with backlighting based on light-emitting diodes
KR101877598B1 (en) 2011-10-14 2018-07-11 메디뮨 리미티드 Pyrrolobenzodiazepines and conjugates thereof
NZ702195A (en) 2012-05-21 2016-09-30 Genentech Inc Anti-ly6e antibodies and immunoconjugates and methods of use
US9492566B2 (en) 2012-12-13 2016-11-15 Immunomedics, Inc. Antibody-drug conjugates and uses thereof
WO2014100762A1 (en) 2012-12-21 2014-06-26 Biolliance C.V. Hydrophilic self-immolative linkers and conjugates thereof
WO2015023355A1 (en) 2013-08-12 2015-02-19 Genentech, Inc. 1-(chloromethyl)-2,3-dihydro-1h-benzo[e]indole dimer antibody-drug conjugate compounds, and methods of use and treatment
US10533058B2 (en) 2013-12-16 2020-01-14 Genentech Inc. Peptidomimetic compounds and antibody-drug conjugates thereof
RU2689388C1 (en) 2013-12-16 2019-05-28 Дженентек, Инк. Peptidomimetic compounds and their conjugates of antibodies with drugs
AU2014364927A1 (en) 2013-12-16 2016-07-07 Genentech, Inc. Peptidomimetic compounds and antibody-drug conjugates thereof
WO2016081643A1 (en) 2014-11-19 2016-05-26 Genentech, Inc. Anti-transferrin receptor antibodies and methods of use
WO2017201449A1 (en) * 2016-05-20 2017-11-23 Genentech, Inc. Protac antibody conjugates and methods of use
US20200038378A1 (en) 2018-04-01 2020-02-06 Arvinas Operations, Inc. Brm targeting compounds and associated methods of use
WO2019195201A1 (en) 2018-04-01 2019-10-10 Arvinas Operations, Inc. Brm targeting compounds and associated methods of use
EP3870235A1 (en) * 2018-10-24 2021-09-01 F. Hoffmann-La Roche AG Conjugated chemical inducers of degradation and methods of use
WO2021067606A1 (en) * 2019-10-01 2021-04-08 Arvinas Operations, Inc. Brm targeting compounds and associated methods of use

Also Published As

Publication number Publication date
AU2021312225A1 (en) 2023-02-16
CR20230017A (en) 2023-02-17
BR112023001143A2 (en) 2023-02-14
US20230330249A1 (en) 2023-10-19
CA3188649A1 (en) 2022-01-27
TW202216215A (en) 2022-05-01
AR123019A1 (en) 2022-10-26
EP4185328A1 (en) 2023-05-31
MX2023000888A (en) 2023-02-22
JP2023535409A (en) 2023-08-17
CL2023000193A1 (en) 2023-07-28
WO2022020288A1 (en) 2022-01-27
CO2023000679A2 (en) 2023-01-26
IL299860A (en) 2023-03-01
KR20230042032A (en) 2023-03-27
PE20231104A1 (en) 2023-07-19

Similar Documents

Publication Publication Date Title
AU2015273098B2 (en) Auristatin derivatives and conjugates thereof
CA2852860C (en) Cytotoxic peptides and antibody drug conjugates thereof
AU2013340449B2 (en) Spliceostatin analogs
US20230330249A1 (en) Antibody-conjugated chemical inducers of degradation of brm and methods thereof
CN116999572A (en) Antibody-drug conjugate, pharmaceutical composition and application thereof in preparation of drugs for treating tumors
TW202016081A (en) Small molecule modulators of human sting, conjugates and therapeutic applications
JP2016516035A (en) Cell growth inhibitors and their conjugates
KR20230035690A (en) Anti-egfr antibody drug conjugates
JP2022505450A (en) Conjugated chemical decomposition inducers and usage
JP2023528412A (en) Anti-BCMA antibody-drug conjugates and methods of use
CA3202759A1 (en) Mcl-1 inhibitor antibody-drug conjugates and methods of use
CA3089754A1 (en) Antibody drug conjugates (adcs) with nampt inhibitors
WO2023147328A1 (en) Antibody-conjugated chemical inducers of degradation with hydolysable maleimide linkers and methods thereof
WO2023147329A1 (en) Antibody-conjugated chemical inducers of degradation and methods thereof
WO2023221975A1 (en) Antibody-drug conjugate containing protein degradation agent bioactive compound, method for preparing same, and use thereof
WO2023170608A1 (en) Activators of effector t cells
KR20230154892A (en) Anti-HER2 antibody-drug conjugates and uses thereof
GB2574913A (en) Small molecule modulators of human STING, conjugates and therapeutic applications
NZ624470B2 (en) Cytotoxic peptides and antibody drug conjugates thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 40088636

Country of ref document: HK